Emergence of carbapenem-resistant enterobacterales co-harboring blaOXA-78 and blaOXA-58 from India.

BACKGROUND: Carbapenem-Resistant Enterobacterales (CRE) has been categorized as pathogens of critical priority by World Health organization (WHO) as they pose significant threat to global public health. Carbapenemase production considered as the principal resistance mechanism against carbapenems and with the recent surge and expansion of carbapenemases and its variants among clinically significant bacteria in India, the present study reports expansion blaOXA-78 and blaOXA-58 of in CRE of clinical origin. METHODS: Bacterial isolates were collected from a tertiary referral hospital and identified through VITEK® 2 Compact automated System (Biomerieux, France). Rapidec® Carba NP (Biomerieux, France) was used to investigate carbapenemase production followed by antibiotic susceptibility testing through Kirby-Bauer Disc Diffusion method and agar dilution method. Class D carbapenemase genes were targeted through PCR assay followed by investigation of horizontal transmission of blaOXA-58 and blaOXA-78. Whole genome sequencing was carried out using Illumina platform to investigate the genetic context of blaOXA-58 and blaOXA-78 genes and further characterization of the CRE isolates. RESULTS: The carbapenem-resistant Escherichia coli (BJD_EC456) and Serratia marcescens (BJD_SM81) received during the study from the tertiary referral hospital were isolated from sputum and blood samples respectively. PCR assay followed by whole genome sequencing revealed that the isolates co-harbor blaOXA-58 and blaOXA-78, a variant of blaOXA-51. Horizontal transfer of blaOXA-58 and blaOXA-78 genes were unsuccessful as these genes were located on the chromosome of the study isolates. Transposon Tn6080 was linked to blaOXA-78 in the upstream region while the insertion sequences ISAba26 and ISCfr1 were identified in the upstream and downstream region of blaOXA-58 gene respectively. In addition, both the isolates were co-harboring multiple antibiotic resistance genes conferring clinical resistance towards beta-lactams, aminoglycosides, fluroquinolones, sulphonamides, tetracyclines. BJD_EC180 belonged to ST2437 while BJD_SM81 was of an unknown sequence type. The nucleotide sequences of blaOXA-78 (OQ533021) and blaOXA-58 (OQ533022) have been deposited in GenBank. CONCLUSIONS: The study provides a local epidemiological information regarding carbapenem resistance aided by transposon and insertion sequences associated blaOXA-78 and blaOXA-58 genes associated and warrants continuous monitoring to prevent their further dissemination into carbapenem non-susceptible strains thereby contributing to carbapenem resistance burden which is currently a global concern.

Transcriptional Response of vanB Operon in Staphylococcus aureus Against Vancomycin and Teicoplanin Stress.

Staphylococcus aureus is a global pathogen and is responsible for causing severe life-threatening infections. The current study was designed to investigate transcriptional expression of different core, regulatory, and accessory genes within vanB operon under differential exposure of vancomycin and teicoplanin. Four isolates selected for the study, were confirmed to harbour vanB gene in which three isolates showed MIC breakpoint above 16 µg/ml and one isolate above 8 µg/ml against vancomycin while teicoplanin showed higher MIC breakpoint as compared to vancomycin. Antibiotic susceptibility results showed that these isolates were susceptible towards imipenem and linezolid. Transcriptional expressional analysis of the core gene of vanB operon showed that expression of vanB is increased under vancomycin stress but is inversely proportional to increase in the concentration of the vancomycin while under teicoplanin stress the expression of vanB showed no significant pattern. Similar expressional pattern was found for vanH gene for both the glycopeptides. In case of vanX, expression was significantly increased at 1 µg/ml exposure of vancomycin, however, no pattern could be observed in case of teicoplanin stress. In case of regulatory gene, vanR, significant increase in expression was observed under vancomycin and teicoplanin stress of 1 µg/ml, however vanS, showed significant increase in the expression under 1 µg/ml of vancomycin. The accessory gene, vanY showed marginal increase in expression under both the antibiotic, while in case of vanW, the expressional pattern was found to be inversely proportional to the increasing antibiotic concentration.

Expressional Pattern of psm-mec System in Methicillin-Resistant Staphylococcus aureus Under Oxacillin Stress.

The psm-mec element and other regulatory factors such as sarA, agrA, and RNAIII are responsible for maintaining the genetic framework for enhanced virulence of MRSA. psm-mec is found predominantly in the staphylococcal cassette chromosome (SCCmec). sarA, agrA, and RNAIII control gene expression to facilitate adaptation in certain environment. Genome-wide approaches have shown that expression of virulence factors is frequently regulated at transcriptional, translational level, and mRNA degradation level. In this study, transcriptional responses of psm-mec gene in accordance with other regulatory factors sarA, agrA, and RNAIII were observed under normal conditions as well as when exposed to 2 µg/ml and 6 µg/ml of oxacillin stress. One-way t-test was carried out for analysing RQ values obtained through real-time PCR. This study showed downregulation of psm-mec gene and upregulation of other regulatory genes at lower concentration of oxacillin. However, this was reverse when exposed against higher concentration of oxacillin. It was observed from the study that the expression of virulence factors were dependent on each other under different concentration of oxacillin. Thus, this study highlights that psm-mec, sarA, agrA, and RNAIII gene are under direct control of antibiotic pressure in a concentration-dependent manner.

Expansion of acquired 16S rRNA methytransferases along with CTX-M-15, NDM and OXA-48 within three sequence types of Escherichia coli from northeast India.

BACKGROUND: This study aimed to identify ten different 16S rRNA methyltransferase genes (rmtA, rmtB, rmtC, rmtD, armA, rmtF, npmA, rmtH, rmtE and rmtG) and their coexisting ESBL and carbapenemase with the emergence of three E.coli clones within a single study centre. METHODS: A total of 329 non-duplicate E.coli isolates were studied to detect the presence of 16S rRNA methyltransferases along with ß-lactamases (TEM, SHV, OXA, VEB, GES, PER,CTX-M types, NDM, OXA-48,VIM, IMP and KPC) using PCR assay. Horizontal transferability were validated by transformation and conjugation analysis. Plasmid incompatibility typing and MLST analysis was also performed. RESULTS: A total of 117 isolates were found to be resistant to at least one of the aminoglycoside antibiotics. It was observed that 77 (65.8%) were positive for 16S rRNA methyltransferases. Among them thirty nine isolates were found to harbour only blaCTX-M-15, whereas combination of genes were observed in three isolates (blaVEB+ blaCTX-M-15 in 2 isolates and blaPER + blaCTX-M-15 in 1 isolate). blaNDM and blaOXA-48 like genes were found in 23 and 9 isolates, respectively. All the resistance genes were conjugatively transferable, and incompatibility typing showed multiple 16S rRNA methyltransferase genes were originated from a single Inc. I1 group. MLST analysis detected 3 clones of E.coliST4410, ST1341 and ST3906. CONCLUSION: The present study identified emergence of three clones of E.coli, resistant to aminoglycoside -cephalosporin- carbapenem. This warrants immediate measures to trace their transmission dynamics in order to slow down their spread in clinical setting.

Correction to: Expansion of acquired 16S rRNA methytransferases along with CTX-M-15, NDM and OXA-48 within three sequence types of Escherichia coli from northeast India.

An amendment to this paper has been published and can be accessed via the original article.

AcrAB-TolC efflux pump system plays a role in carbapenem non-susceptibility in Escherichia coli.

BACKGROUND: Efflux pump mediated antibiotic resistance is an unnoticed and undetected mechanism in clinical microbiology laboratory. RND efflux systems are known for aminoglycoside and tetracycline resistance whereas their role in carbapenem non-susceptibility is not established. The study was undertaken to investigate the role of efflux pump in providing resistance against carbapenems and their response against concentration gradient carbapenem stress on the transcriptional level of the AcrAB gene in the clinical isolates of Escherichia coli from a tertiary referral hospital of Northeast India. RESULTS: Out of 298 non-susceptible Escherichia coli isolates 98 isolates were found to have efflux pump mediated carbapenem non-susceptibility. Among them thirty-five were non carbapenemase producers and their expressional levels were verified using qRT-PCR under concentration gradient carbapenem stress. In this study, a strong correlation between ertapenem resistance and AcrA overexpression was observed which has not been reported previously. Further, it was observed that imipenem stress increased AcrB expression in Escherichia coli which holds the novelty of this study. Additionally, the transcription of AcrR was insistently increased which is much higher than the transcriptional level of AcrA under concentration gradient carbapenem stress condition. CONCLUSION: The study established that AcrAB pump is a relevant antibiotic resistance determinant in bacterial pathogen, has an important role in developing resistance against carbapenem group of antibiotics.

Detection and molecular typing of methicillin-resistant Staphylococcus aureus from northeastern part of India.

BACKGROUND: In Staphylococcus aureus, methicillin resistance is exhibited by modifications in penicillin-binding protein that minimises the binding affinity to beta-lactam antibiotics. The present study investigated the occurrence of methicillin-resistant S. aureus (MRSA) in community-acquired infections, that is, community-acquired MRSA (CA-MRSA) and in-hospital-acquired infections, that is, hospital-acquired MRSA (HA-MRSA) from Northeast India. METHODS: A total of 197 consecutive non-duplicate isolates were collected from Silchar Medical College and Hospital and other private diagnostic laboratories. The isolates were confirmed to be S. aureus at our centre. All isolates were subjected to antibiotic susceptibility testing and were screened for methicillin resistance using cefoxitin disc test. All MRSA were subjected to Polymerase Chain Reaction (PCR) assay for detection of mecA and mecC genes. DNA fingerprinting was performed for determining clonal diversity. RESULTS: Seventy-one isolates of 127 confirmed S. aureus were found to be methicillin resistant by screening test. mecA gene was detected in 43 isolates, and none of the isolates were positive for mecC gene. Linezolid and teicoplanin showed better activity with susceptibility pattern being 83.6% and 72.44%, respectively, whereas 66.14% were sensitive to vancomycin. Other antibiotic showed low level of activity. Pulsed Field Gel Electrophoresis (PFGE) showed 14 different banding patterns that suggest isolates were of different clonal types. CONCLUSION: mecA was responsible for methicillin resistance in majority of strains. Polyclonal spread of MRSA infection in the study area indicates its diverse origin and possible lateral transfer. Thus, this study is of clinical interest in terms of selection of proper antimicrobial chemotherapy and infection control management.

Occurrence of bla DHA-1 mediated cephalosporin resistance in Escherichia coli and their transcriptional response against cephalosporin stress: a report from India.

BACKGROUND: Treatment alternatives for DHA-1 harboring strains are challenging as it confers resistance to broad spectrum cephalosporins and may further limit treatment option when expressed at higher levels. Therefore, this study was designed to know the prevalence of DHA genes and analyse the transcription level of DHA-1 against different ß-lactam stress. METHODS: Screening of AmpC ß-lactamase phenotypically by modified three dimensional extract method followed by Antimicrobial Susceptibility and MIC determination. Genotyping screening of ß-lactamase genes was performed by PCR assay followed by their sequencing. The bla DHA-1 transcriptional response was evaluated under different cephalosporin stress by RT PCR. Transferability of bla DHA gene was performed by transformation and conjugation and plasmid incompatibility typing, DNA fingerprinting by enterobacterial repetitive intergenic consensus sequences PCR. RESULTS: 16 DHA-1 genes were screened positive from 176 Escherichia coli isolates and primer extension analysis showed a significant increase in DHA-1 mRNA transcription in response to cefotaxime at 8 µg/ml (6.99 × 102 fold), ceftriaxone at 2 µg/ml (2.63 × 103 fold), ceftazidime at 8 µg/ml (7.06 × 103 fold) and cefoxitin at 4 µg/ml (3.60 × 104 fold) when compared with untreated strain. These transcription data were found significant when analyzed statistically using one way ANOVA. Four different ESBL genes were detected in 10 isolates which include CTX-M (n = 6), SHV (n = 4), TEM (n = 3) and OXA-10 (n = 1), whereas, carbapenemase gene (NDM) was detected only in one isolate. Other plasmid mediated AmpC ß-lactamases CIT (n = 9), EBC (n = 2) were detected in nine isolates. All DHA-1 genes detected were encoded in plasmid and incompatibility typing from the transformants indicated that the plasmid encoding bla DHA-1 was carried mostly by the FIA and L/M Inc group. CONCLUSION: This study demonstrates the prevalence of DHA-1 gene in this region and highlights high transcription of DHA-1 when induced with different ß-lactam antibiotics. Therefore, cephalosporin treatment must be restricted for the patients infected with pathogen expressing this resistance determinant.

Integron-borne transmission of VEB-1 extended-spectrum ß-lactamase in Pseudomonas aeruginosa in a tertiary care hospital in India.

A total 14 clinical isolates of Pseudomonas aeruginosa that produced VEB-1 and were susceptible only to polymyxin B were recovered from hospitalized patients. VEB-1 was located within variable regions of the class 1 integron, flanked by resistant genes, and was horizontally transferable as well as carried within the IncP-type plasmid. We conclude that the IncP-type plasmid is responsible for the horizontal transmission of VEB-1-mediated expanded-spectrum cephalosporin resistance in this medical center.

Concentration dependent exposure of vancomycin and teicoplanin induces vanG regulon in Staphylococcus aureus.

Therapeutic options for staphylococcus infections have been raised due to the emergence of VISA and VRSA. Six isolates of Staphylococcus aureus of clinical origin which were previously confirmed to carry vanG were selected for this study. Antimicrobial susceptibility was performed by disc diffusion method. Transcriptional expression of vanG and vanSG showed down regulation against vancomycin and teicoplanin but expression was increased with increasing concentration of antibiotics. vanUG, vanRG showed up regulation against glycopeptide exposure. The present study underscored that expression of vanG and its regulatory gene operons are dependent on concentration of vancomycin and teicoplanin exposure in S.aureus.

Elevated expression of rsmI can act as a reporter of aminoglycoside resistance in Escherichia coli using kanamycin as signal molecule.

We aimed to design and analyse expressional response of endogenous and exogenous 16S rRNA methyl transferase genes under sub inhibitory concentration stress of different clinically relevant aminoglycoside antibiotics in Escherichia coli to identify an endogenous marker. One hundred twenty nine aminoglycoside resistant E. coli of clinical origin were collected for detection of 16S rRNA methyl transferase genes by PCR assay and each gene type was cloned within E. coli JM107. Parent isolates were subjected to plasmid elimination by SDS treatment. Expression analysis of both acquired and endogenous 16S rRNA methyl transferase genes were performed by quantitative real-time PCR in clones and parent isolates under aminoglycoside stress (4 mg/L). Majority of the isolates were harbouring rmtC (35/129), followed by rmtB (32/129), rmtA (21/129), rmtE (13/129), armA (11/129) rmtF (9/129) and rmtH (8/129). Plasmid was successfully eliminated for all the isolates with 6% of SDS. Expression analysis indicates that kanamycin, tobramycin and netilmicin stress could increase the expression of 16S rRNA methyltransferese genes. In the presence of kanamycin stress the expression of rsmI was consistently elevated for all the wild type isolates and clones tested. Except for isolates harbouring rmtB and rmtC expression of rsmE and rsmF was increased in the presence of all aminoglycosides. For all the cured mutants it was apparently observed that expression of endogenous methyl transferases were marginally increased. Elevated expression of constitutive rsmI can be used as a potential biomarker for detection of acquired 16S rRNA methyl transferase mediated aminoglycoside resistance by using sub inhibitory concentration of kanamycin as signal molecule.

Escherichia coli harbouring strAB with reduced susceptibility towards gentamicin and amikacin: a single centre study from India.

In this study we report the presence of streptomycin resistance gene strAB within clinical isolates of Escherichia coli where streptomycin is not used to treat Gram-negative infections. In total, 135 E. coli isolates were obtained for the study. PCR based detection of strAB was performed in the study isolates followed by assessment of horizontal transferability. Cloning of strAB was done in laboratory strain E. coli DH5α. Pre-cloning and post-cloning susceptibility of the strain was done for assessment of acquired resistance. Among tested isolates, 89 were found to harbour strAB and it was encoded within a IncI1 type plasmid. Cloning experiments revealed the strAB gene showed unusual non-susceptibility towards amikacin and gentamicin. The study highlighted that strAB, which has a role in streptomycin resistance, may also have a role in reduced susceptibility towards gentamicin and amikacin within a clinical setting.

Transcriptional analysis of prsA and vraTS regulatory system in methicillin resistant Staphylococcus aureus against oxacillin stress.

PURPOSE: The prsA and vraTSR regulatory systems play a unique role in methicillin resistance by modifying the peptidoglycan cell wall PBP2 and involving cell wall stress response in Staphylococcus aureus. This study was designed to observe the transcriptional response of prsA and vraTSR system under oxacillin stress in S.aureus. METHODS: In this study, three clinical isolates of Staphylococcus aureus and a laboratory strain were examined. All the isolates were tested for mecA gene by PCR assay and were also tested for prsA, vraT, vraS and vraR gene. The transcriptional responses of the prsA gene along with the vraTSR regulatory system in these isolates was observed under normal conditions and exposed to 2 â€‹µg/ml and 4 â€‹µg/ml of oxacillin stress by quantitative real-time PCR assay. RESULTS: The result of transcriptional analysis confirmed that under oxacillin stress, the expressions of vraS and vraT are increased with the increase in the concentration of oxacillin. However, prsA has shown no significant expression under oxacillin stress. CONCLUSION: Although prsA did not show any specific expressional pattern, the study highlights the role of vraS and vraT regulatory system in conferring a methicillin-resistant phenotype when exposed to subinhibitory concentrations of oxacillin, which could act as a potential target for the next-generation antimicrobials.

Colistin exposure enhances expression of eptB in colistin-resistant Escherichia coli co-harboring mcr-1.

Colistin resistance has increased due to the increasing and inappropriate use of this antibiotic. The mechanism involves modification of lipid A with phosphoethanolamine (PEtN) and/or 4-amino-4deoxy-L-arabinose (L-Ara4N). EptA and eptB catalyze the transfer of phosphoethanolamine to lipid A. In this study, gene network was constructed to find the associated genes related to colistin resistance, and further in vitro validation by transcriptional analysis was performed. In silico studies showed that eptB gene is a highly interconnected node in colistin resistance gene network. To ascertain these findings twelve colistin-resistant clinical isolates of Escherichia coli were selected in which five were harboring the plasmid-mediated mcr-1. Screening for colistin resistance was performed by broth microdilution (BMD) method and Rapid polymyxin NP test. PCR confirmed the presence of the eptA and eptB genes in all isolates and five isolates were harboring mcr-1. Transcriptional expression in five isolates harboring mcr-1, showed an enhanced expression of eptB when exposed under sub-inhibitory colistin stress. The present study for the first time highlighted genetic interplay between mcr-1 and eptA and eptB under colistin exposure.

Antimicrobial resistance in patients with suspected urinary tract infections in primary care in Assam, India.

OBJECTIVES: We investigated the prevalence and diversity of antimicrobial resistance in bacteria isolated from urine samples of community-onset urinary tract infection (UTI) patients in southern Assam, India. METHODS: Freshly voided midstream urine samples were collected from patients attending primary healthcare centres, with the patients' epidemiological data also recorded. Species identification was confirmed using a VITEK 2 compact automated system. Phenotypic confirmation of ESBLs was performed using the combined disc diffusion method (CLSI 2017) and carbapenemase production was phenotypically characterized using a modified Hodge test. Common ESBLs and carbapenem-resistance mechanisms were determined in Escherichia coli isolates using PCR assays. Incompatibility typing of the conjugable plasmids was determined by PCR-based replicon typing; the phylotypes and MLSTs were also analysed. RESULTS: A total of 301 (59.7%) samples showed significant bacteriuria along with symptoms of UTI and among them 103 isolates were identified as E. coli of multiple STs (ST3268, ST3430, ST4671 and others). Among them, 26.2% (27/103) were phenotypically ESBL producers whereas 12.6% (13/103) were carbapenemase producers. This study describes the occurrence of diverse ESBL genes-bla CTX-M-15, bla SHV-148, bla PER-1 and bla TEM-and two E. coli isolates carrying the bla NDM-1 carbapenemase gene. ESBL genes were located within transconjugable plasmids of IncP and IncF type whereas bla NDM-1 was carried in an IncFrepB type plasmid. CONCLUSIONS: This study illustrates the high rate of MDR in E. coli causing UTI in primary care in rural Assam. UTIs caused by ESBL- or MBL-producing bacteria are very difficult to treat and can often lead to treatment failure. Thus, future research should focus on rapid diagnostics to enable targeted treatment options and reduce the treatment failure likely to occur with commonly prescribed antibiotics, which will help to combat antimicrobial resistance and the burden of UTIs.

An insight into analysis and elimination of plasmids encoding metallo-ß-lactamases in Pseudomonas aeruginosa.

OBJECTIVES: The aim of this study was to characterise metallo-ß-lactamase (MBL)-harbouring plasmids, their change in copy number in respect to different antibiotic pressure, and the efficiency of different curing agents in eliminating these resistance plasmids from nosocomial Pseudomonas aeruginosa isolates. METHODS: Plasmids were extracted from four isolates harbouring blaNDM-1 or blaVIM-2 under four different concentrations of imipenem, meropenem, ertapenem, aztreonam and cefotaxime. Quantitative real-time PCR was performed to analyse the change in plasmid copy number under these different conditions. The effect of different physical and chemical curing agents in elimination of plasmids carrying blaNDM-1 and blaVIM-2 was examined, with meropenem resistance used as a selectable marker. RESULTS: Conjugatively transferable MBL genes (blaNDM-1 and blaVIM-2) carried on plasmids were found to be highly stable. Sodium dodecyl sulfate (SDS) was the most effective agent in eliminating these resistance plasmids. The change in copy number of the blaNDM-1-encoding plasmid was found to be similar to the blaVIM-2-encoding plasmid, with a single exception under cefotaxime pressure. CONCLUSION: The spread of multidrug resistance plasmids has been noted as a key factor associated with increasing carbapenem resistance. Successful curing of resistance plasmids can reverse the bacterial phenotype back to susceptible. This study revealed that different antibiotic pressure induces a change in copy number of MBL-encoding plasmids. SDS can be successfully used as an eliminating agent for these resistance determinants, although therapeutic application of this agent is not possible due to its high toxicity and mutagenic nature.

Transcriptional response of mar, sox and rob regulon against concentration gradient carbapenem stress within Escherichia coli isolated from hospital acquired infection.

OBJECTIVE: The present study was carried out to investigate the transcriptional response of marA (Multiple antibiotic resistance A gene), soxS (Superoxide S gene) and rob (Right-origin-binding gene) under carbapenem stress. RESULTS: 12 isolates were found over-expressing AcrAB-TolC efflux pump system and showed reduced expression of OmpF (Outer membrane porin) gene were selected for further study. Among them, over expression of marA and rob was observed in 7 isolates. Increasing pattern of expression of marA and rob against meropenem was observed. The clones of marA and rob showed reduced susceptibility towards carbapenems.

Use of fluorescence foldscope as an effective tool for detection of biofilm formation in Pseudomonas aeruginosa.

Purpose: Pseudomonas aeruginosa is an opportunistic pathogen with biofilm-forming ability, by the virtue of which they can evade the immune response and antimicrobial chemotherapy. Several methods have been designed for the detection of biofilms but require sophisticated instrumentation and expertise. The present study, therefore, used an improvised device, 'fluorescence foldscope' which is an origami-based fluorescence microscope as an easy and effective tool to detect biofilm formation. Methodology: Three representatives of P. aeruginosa of clinical origin were taken for the study along with two reference strains PA01 and ATCC27853. The strains were cultured in Luria Bertani (LB) broth with and without carbapenem (imipenem and meropenem) and cephalosporin (ceftazidime, cefotaxime and ceftriaxone) pressure, respectively. The cultures were diluted to 1:100 in LB; seeded with sterile glass slides at 90° angle and incubated for 5 consecutive days. The slides were observed with fluorescence foldscope. Results: Fluorescence emission was observed in two test isolates CD1 and CD2 at 48 and 72 h, respectively, whereas no fluorescence was observed in CD3. The fluorescence observed in the isolates was not affected by 2 µg/ml carbapenem pressure, while with 2 µg/ml ceftazidime stress, a change in fluorescence was observed in CD2 in comparison to the fluorescence observed under normal growth condition. Conclusion: Fluorescence foldscopy is an effective and reliable tool for the detection of biofilm formation in clinical isolates of P. aeruginosa under different laboratory conditions. Biofilm-forming P. aeruginosa worsens the medical condition and is difficult to eradicate. The present study came up with an effective and reliable tool for the detection of biofilm formation in clinical isolates of P. aeruginosa.

Pondicherry declaration on the identification and detection of Entamoeba histolytica.

Stake holders meet on "Identification and Detection of Entamoeba histolytica" was conducted on July 21, 2019 at Sri Balaji Vidyapeeth Deemed-to-be-University, Pondicherry. This programme was of national importance, since the amoebiasis is being increasingly reported from different parts of India because of poor socioeconomic conditions and sanitation levels. Experts in amoebiasis research across India attended this meeting. This meeting was conducted with an objective to frame the guidelines on the identification and detection of E. histolytica with reference to conventional diagnostic methods and molecular diagnosis targeting appropriate genes of E. histolytica. The recommendations of the panel were released as declaration on the diagnosis of amoebiasis and were circulated to various administrative and scientific bodies in India as reference policy document on the diagnosis of amoebiasis.