Recording and Reporting of Antimicrobial Resistance (AMR) Priority Variables and Its Implication on Expanding Surveillance Sites in Nepal: A CAPTURA Experience.

Data on antimicrobial resistance (AMR) from sites not participating in the National AMR surveillance network, conducted by National Public Health Laboratory (NPHL), remain largely unknown in Nepal. The "Capturing Data on Antimicrobial Resistance Patterns and Trends in Use in Regions of Asia" (CAPTURA) assessed AMR data from previously untapped data sources in Nepal. A retrospective cross-sectional data review was carried out for the AMR data recorded between January 2017 and December 2019 to analyze AMR data from 26 hospital-based laboratories and 2 diagnostic laboratories in Nepal. Of the 56 health facilities initially contacted to participate in this project activity, 50.0% (28/56) signed a data-sharing agreement with CAPTURA. Eleven of the 28 hospitals were AMR surveillance sites, whereas the other 17, although not part of the National AMR surveillance network, recorded AMR-related data. Data for 663 602 isolates obtained from 580 038 patients were analyzed. A complete record of the 11 CAPTURA priority variables was obtained from 45.5% (5/11) of government hospitals, 63.6% (7/11) of private hospitals, and 54.6% (6/11) of public-private hospitals networked with NPHL for AMR surveillance. Similarly, 80% (8/10) of clinics and 54.6% (6/11) of laboratories outside the NPHL network recorded complete data for the 10 Global Antimicrobial Resistance and Use Surveillance System (GLASS) priority variables and 11/14 CAPTURA priority variables. Retrospective review of the data identified areas requiring additional resources and interventions to improve the quality of data on AMR in Nepal. Furthermore, we observed no difference in the priority variables reported by sites within or outside the NPHL network, thus suggesting that policies could be made to expand the surveillance system to include these sites without substantially affecting the government's budget.

Has Data Quality of an Antimicrobial Resistance Surveillance System in a Province of Nepal Improved between 2019 and 2022?

An operational research study was conducted in 2019 to assess the quality of data submitted by antimicrobial resistance (AMR) surveillance sites in the Bagmati Province of Nepal to the National Public Health Laboratory for Global Antimicrobial Resistance and Use Surveillance System (GLASS). Measures were implemented to enhance the quality of AMR surveillance by strengthening capacity, improving infrastructure, implementing data sharing guidelines, and supervision. The current study examined reports submitted by surveillance sites in the same province in 2022 to assess whether the data quality had improved since 2019. The availability of infrastructure at the sites was assessed. Of the nine surveillance sites in the province, seven submitted reports in 2022 versus five in 2019. Completeness in reporting improved significantly from 19% in 2019 to 100% in 2022 (p < 0.001). Timely reports were received from two sites in 2019 and only one site in 2022. Specimen-pathogen consistency in accordance with the GLASS guidelines for urine, feces, and genital swab specimens improved, with ≥90% consistency at all sites. Overall, the pathogen-antibacterial consistency improved significantly for each GLASS priority pathogen. The study highlights the importance of dedicated infrastructure and institutional arrangements for AMR surveillance. Similar assessments covering all provinces of the country can provide a more complete country-wide picture.

Integrative Digital Tools to Strengthen Data Management for Antimicrobial Resistance Surveillance in the "One Health" Domain in Nepal.

Antimicrobial resistance (AMR) is increasing and represents one of the greatest public health challenges of our time, accounting for considerable morbidity and mortality globally. A "One Health" surveillance strategy, which integrates data concerning the resistant organisms circulating in humans, animals, and the environment, is required to monitor this issue and enable effective interventions. The timely collection, processing, analysis, and reporting of AMR surveillance data are necessary for the effective delivery of the information generated from such surveillance. Nepal has greatly improved its surveillance activities through a network of human and animal health laboratories; however, the data reported by sentinel laboratories are often inconsistent, incomplete, and delayed, causing challenges in terms of data cleaning, standardization, and visualization on a national level. To overcome these issues, innovative methods and procedures have been adopted in Nepal, with the development and customization of digital tools that reduce the human time and effort spent on data cleaning and standardization, with concomitant improvements in the accuracy of data. These standardized data can be uploaded to the district health information system 2 (DHIS2) One Health AMR surveillance portal, enabling the generation of reports that will help decision-makers and policy planners to combat the global problem of AMR.

Cryptococcal meningitis in people living with human immunodeficiency virus in Nepal: Perspectives from resource limited setting.

Early diagnosis of cryptococcal meningitis among people living with HIV (PLHIV) is crucial for its therapeutic success. The objective of this study was to diagnose cryptococcal meningitis in PLHIV cases using the available laboratory techniques for its confirmation in resource limited setting. This cross-sectional prospective study was conducted among 72 PLHIV with clinical suspicion of meningitis. Each cerebrospinal fluid (CSF) sample received at the National Public Health Laboratory, Kathmandu was processed for India ink staining, cryptococcal antigen lateral flow assay, and fungal culture following standard protocols. The laboratory-confirmed cryptococcal meningitis cases were between 24 and 69 years of age (median age 39 years) with 87.5% (12/14) of cases being male. Cryptococcus was detected in 22.22% (16/72) by any of the three tests, 19.44% (14/72) by cryptococcal antigen lateral flow assay, 16.66% (12/72) by India ink staining, and 8.33% (6/72) by culture. High percentage of cryptococcal meningitis among PLHIV warrants early microbiological diagnosis for better case management. Cryptococcal antigen detection immunoassay should be the priority test for laboratory diagnosis of cryptococcal meningitis in PLHIV. Alternatively, very simple and economic India ink staining of CSF specimens could be used in resource limited settings.

Changing pattern of severe manifestations of Plasmodium falciparum and Plasmodium vivax malaria: A retrospective study from Bikaner, India.

BACKGROUND & OBJECTIVES: Previously there were reports from all over India about the changing spectrum of severe manifestations of Plasmodium falciparum malaria. Consequently, the present retrospective study was conducted to compare the severity of malaria caused by P. falciparum and P. vivax during 2007-08 and 2017-18. METHODS: The present study was conducted on the patients admitted with severe malaria in a classified malaria ward of a tertiary care hospital in Bikaner, Rajasthan (Northwest India) during 2007-08 and 2017-18. It included adult patients of both sexes belonging to all age groups. The diagnosis was done by peripheral blood film (PBF), rapid diagnostic test (RDT), and validated by polymerase chain reaction (PCR). All patients were treated with intravenous oral quinine. The specific individual malaria complications registered in 2007-08 and 2017-18 were treated by following the standard WHO protocol. RESULTS: In 2007-08, severe manifestations caused by P. falciparum were dominated by thrombocytopenia (25.98%) followed by jaundice (24.39%), multi-organ dysfunction (MODS) (16.66%), severe anemia (16.17%), cerebral malaria (5.39%), bleeding manifestation (3.92%) and shock (0.49%). While in the same year, P. vivax associated clinical spectrum of complications were observed to be dominated by thrombocytopenia (26.47%) followed by jaundice (25.00%), MODS (14.70%), severe anemia (5.88%), cerebral malaria (5.88%), renal failure (4.41%), bleeding manifestation (2.45%), shock (0.98%) and acute respiratory distress syndrome (ARDS) (0.49%). However, in 2017-18, the clinical spectrum of malaria complications caused by both species has changed. Relative to P. falciparum infections, P. vivax individual complications like thrombocytopenia (51.78%) (p<0.001) followed by jaundice (19.13%) (p<0.001) and severe anemia (4.22%) (p<0.05) were found to have increased significantly. INTERPRETATION & CONCLUSION: Over the last decade there is an apparent spatial and temporal shift in the clinical manifestations of severe malaria caused by the both Plasmodium species. As evident from the patient's data from 2007-08 and 2017-18, the severity is more inclined towards Plasmodium vivax than Plasmodium falciparum malaria. Moreover, individual P. falciparum-associated complications were decreased significantly in the Bikaner region of Rajasthan, India.

Detection of Beta-Lactamases (ESBL and MBL) Producing Gram-Negative Pathogens in National Public Health Laboratory of Nepal.

Background: Bacterial resistance to antibiotics has increased in recent years. Resistance to ß-lactams in Gram-negative bacteria has been reported to be associated with extended spectrum beta-lactamases and metallo-beta-lactamases. This study was aimed at determining the distribution and antibiotic susceptibility patterns of Gram-negative pathogens producing extended spectrum beta lactamases and metallo-beta lactamases. Method and Methodology. This cross-sectional study was conducted at the National Public Health Laboratory during a period of six months. All clinical specimens were obtained and processed for the identification of Gram-negative pathogens by culture, morphological, and biochemical tests. Antibiotic susceptibility testing of the isolates was performed by the Kirby Bauer disc diffusion and the isolates were tested for ESBL and MBL by the combined disk method. Results: Out of 4266 clinical specimens, 197 (4.6%) were found to be Gram-negative bacterial isolates. 47 (23.9%) isolates were ESBL producers. The most predominant organisms were Escherichia coli (53%), Klebsiella pneumonia (23%), and Pseudomonas spp. (13%). 16 (8.2%) were positive for MBL producers, and 6(3.1%) were both ESBL and MBL producers. The MBL activity was seen in E. coli (38%), followed by Pseudomonas spp. (31%), and K. pneumoniae (19%). The ESBL producers showed a higher degree of sensitivity towards imipenem and amikacin, followed by piperacillin tazobactam. MBL producers showed sensitivity towards amikacin only. Conclusion: The prevalence of ESBL and MBL producing Gram-negative bacteria was found to be high in bacterial infections in Nepal. Routine laboratory testing for ESBL and MBL is needed in order to optimize antibiotic management and reduce the risk of spread of infections caused by ESBL and MBL producers.

Meningococcal Carriage among Household Contacts of Patients with Invasive Meningococcal Disease in Kathmandu, Nepal: A Longitudinal Study.

Because asymptomatic carriers are key source of transmission, information on meningococcal carriage in the community provides a scientific basis for appropriate preventive/control strategies. This longitudinal study (January 2017-December 2019) aimed to estimate carriage rate of meningococci among household contacts of meningococcal meningitis cases within Kathmandu Valley, Nepal. Throat swab samples were collected at first visit from each person in households, twice a month for up to 2 months and subsequently on a monthly basis for a further 4 months. Altogether, 1125 throat samples were processed by conventional culture for the identification of meningococci. To the best of our knowledge, this is the first longitudinal study on meningococcal carriage in Nepal. The meningococcal carriage rate among household contacts was 15%. All carriers were aged 19 years or older. There was no statistically significant gender difference. The duration of carriage was 60 days. Twenty of 36 isolates belonged to serogroup A, and 16 were non-serogroupable (NG). Serogroups isolated from the same individuals did not change within the follow-up period. All meningococcal isolates over the past 38 years in Nepal that have been reported in previous studies have belonged to serogroup A. The detection of NG meningococcal isolates in apparently healthy household contacts clearly indicates the importance of vigilance through surveillance and periodic in-depth studies.

Detection of Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae in Culture Negative Cerebrospinal Fluid Samples from Meningitis Patients Using a Multiplex Polymerase Chain Reaction in Nepal.

The rapid identification of bacteria causing meningitis is crucial as delays in the treatment increase mortality rate. Though considered as the gold standard for the laboratory diagnosis of bacterial meningitis, culture might give false negative results in a case of patients under antibiotics prior to lumbar puncture. This study aimed to detect Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae by a multiplex polymerase chain reaction (PCR) in culture-negative cerebrospinal fluid samples collected from clinically suspected meningitis cases attending different hospitals in Kathmandu, Nepal from January 2017 to December 2019. S. pneumoniae, N. meningitidis and H. influenzae were detected in 8.59% (33/384) of the specimens by PCR and 7.55% (29/384) of the specimens by culture. Correlation between culture and PCR of the same sample was good (Spearman's rho correlation coefficient = 0.932). However, the difference in positivity between culture and PCR was statistically not significant (p value > 0.05). In four specimens, culture could not detect any of the targeted bacteria whereas PCR could detect presence of H. influenzae. PCR increases the diagnostic yield for bacterial meningitis. PCR may be considered as an adjunctive test for establishing the cause of infection in culture negative clinically suspected meningitis cases.

Quality Assessment of an Antimicrobial Resistance Surveillance System in a Province of Nepal.

Antimicrobial resistance (AMR) is a global problem, and Nepal is no exception. Countries are expected to report annually to the World Health Organization on their AMR surveillance progress through a Global Antimicrobial Resistance Surveillance System, in which Nepal enrolled in 2017. We assessed the quality of AMR surveillance data during 2019-2020 at nine surveillance sites in Province 3 of Nepal for completeness, consistency, and timeliness and examined barriers for non-reporting sites. Here, we present the results of this cross-sectional descriptive study of secondary AMR data from five reporting sites and barriers identified through a structured questionnaire completed by representatives at the five reporting and four non-reporting sites. Among the 1584 records from the reporting sites assessed for consistency and completeness, 77-92% were consistent and 88-100% were complete, with inter-site variation. Data from two sites were received by the 15th day of the following month, whereas receipt was delayed by a mean of 175 days at three other sites. All four non-reporting sites lacked dedicated data personnel, and two lacked computers. The AMR surveillance data collection process needs improvement in completeness, consistency, and timeliness. Non-reporting sites need support to meet the specific requirements for data compilation and sharing.

Pigments, ascorbic acid, total polyphenols and antioxidant capacities in deetiolated barley (Hordeum vulgare) and wheat (Triticum aestivum) microgreens.

The study was aimed to evaluate if deetiolation of barley and wheat microgreens after cultivaton in dark (for 5, 7 and 9 days) can enhance the contents of pigments, ascorbic acid, polyphenols, and equivalent antioxidant capacities (EAC) (measured by DPPH and FRAP assay) in correlation to other. Chlorophylls and carotenoids were higher in microgreens that were exposed more to daylight. In contrast, ascorbic acid, polyphenols and EAC of microgreens could be enhanced by 5-7 days of etiolation. However, prolonged etiolation reduced overall antioxidant capacities of microgreens. All evaluated parameters could be satisfactorily represented by regression expressions for the given number of days of etiolation and growth. The ascorbic acid and total carotenoids content had higher correlations with total chlorophyll contents, while the antioxidant capacities were highly correlated to total polyphenols content. The study confirms the potential of deetiolated cultivation of microgreens to enhance selective phytochemicals content and EAC of microgreens.

Comparison of acridine orange fluorescent microscopy and gram stain light microscopy for the rapid detection of bacteria in cerebrospinal fluid.

OBJECTIVE: Bacterial meningitis is a life threatening condition that requires prompt recognition and treatment. Currently, Gram stain is widely used for the microscopic detection of bacterial pathogens in cerebrospinal fluid (CSF). In Nepal, fluorescent microscopes have been installed in laboratories as a part of the National tuberculosis control program. However, information on the utility of the acridine orange (AO) stain for the direct detection of bacteria in CSF samples in Nepal is not available. Therefore, this study aims to compare Gram stain and AO stain for the rapid detection of bacterial pathogens in CSF of clinically suspected meningitis cases in Kathmandu, Nepal. RESULTS: Bacterial pathogens were detected in 9.30% (36/387) by either of the three tests, 9.04% (35/387) by AO stain, 8.27% (32/387) by culture and 6.46% (25/387) by Gram's stain. Considering culture as a gold standard, the sensitivity of AO stain was higher than Gram stain. The specificity of AO stain was 98.87%. Detection and differentiation of the bacteria was much clear in AO staining than Gram staining. AO is a better alternative to Gram stain in the rapid detection of bacterial pathogens in CSF in the setting where fluorescent microscope is available.

Changing epidemiology and antimicrobial resistance in Vibrio cholerae: AMR surveillance findings (2006-2016) from Nepal.

BACKGROUND: In Nepal, cases of Cholera occur annually either as sporadic or as outbreaks claiming the lives of many in rural areas. The present study is a laboratory based surveillance which aims to analyze the changing epidemiology and antimicrobial susceptibility trend of V. cholerae strains isolated or referred to National Public Health Laboratory (NPHL) over a period of 11 years (2006-2016). METHODS: Specimens of fresh stool /rectal swab either received at sentinel sites or NPHL were processed following standard microbiological techniques. Suspected colonies on selective medium were identified using routine biochemical tests and confirmed by serotyping. Antimicrobial susceptibility testing was performed following Kirby Baeur disc diffusion method. RESULTS: Of the 836 confirmed isolates, 87% (728/836) were V.cholerae O1 Ogawa,12% (103/836) were V.cholerae O1 Inaba and only 6 isolates were V.cholerae O1 Hikojima. In 2006 all the Vibrio isolates were of Inaba serotype, followed by all 3 serotypes during 2007.During 2008-2014 only Ogawa serotype was isolated while few cases of Inaba again surfaced in 2015. Resistance to ampicillin decreased from 93% in 2006 to 18% by 2010 and again raised to 100% by 2016.Cotrimoxazole resistance remained at constant range (77-100%).Nalidixic acid resistance was 100% since 2006.Ciprofloxacin and tetracycline resistance emerged in 2007, reached a peak during 2010-2012 and declined to 0 by 2016.Susceptibility to Furazolidone has re-emerged.63.6% of the isolates were Multi drug resistant. CONCLUSION: With changing epidemiology and antibiogram of V.cholerae in Nepal, the present study reflects the importance of continuous monitoring, which could be used by policy makers and health professionals for better management of outbreaks. Decline in tetracycline and ciprofloxacin resistance along with emerging sensitivity to furazolidone shows that these drugs could make an effective comeback in future.

Outbreak Investigation Following the 2015 Earthquake Disaster in Nepal.

BACKGROUND: Infectious disease outbreaks following natural disasters are reported in literature. Outbreaks were documented following natural disasters in many countries including Haiti. Such possibility following 2015 Nepal earthquake was a public health concern. Risk factors needed evaluation by post-disaster outbreak investigation. Hence, present study was undertaken to investigate potentials for such outbreak and to generate evidence for public health intervention. METHODS: The study was conducted between April - May, 2015, with the cooperation of National Public Health Laboratory, Epidemiology and Disease Control Division, Nepal Health Research Council and the Chinese team. Rapid Response Teams visited earthquake affected districts and collected samples for analysis. Syndromic surveillance approach was followed. Samples were collected from syndromic patients under supervision. Those sick prior to earthquake or receiving treatment were excluded. Blood, stool and throat swab samples, as indicated, were collected. Drinking water and food samples including captured live mosquitoes from inhabited areas were obtained for study with the help of EDCD. Laboratory analysis was performed at the NPHL. RESULTS: Total samples were 360 (114 biological, 246 environmental). Salmonella spp. was detected in two and Varicella zoster in one blood sample. Influenza B virus was detected in one throat swab. Rota virus was detected in two, Shigella dysenteriae in one and Salmonella spp. in one stool sample. No pathogen detected in water or food samples. Mosquitoes tested negative for dengue virus. CONCLUSIONS: Post-earthquake outbreak investigated in disaster phase-2. Diarrheal, enteric fever pathogens and Influenza B virus were detected. Environmental samples tested negative for pathogens. Vigilance is necessary for other risk factors.

Comparison of Nasal Colonization of Methicillin-Resistant Staphylococcus aureus in HIV-Infected and Non-HIV Patients Attending the National Public Health Laboratory of Central Nepal.

BACKGROUND: Staphylococcus aureus is a cardinal source of community- and hospital-acquired infection. HIV infection is a well-recognized risk factor for methicillin-resistant S. aureus (MRSA) carriage and infection. Intrinsically developed antibiotic resistance has sharply increased the burden of MRSA which is often associated with morbidity and mortality of the patients. Moreover, nasal carriage of S. aureus plays a significant role in spread of community-associated (CA) S. aureus infections. METHODS: This study was conducted from June 2016 to December 2016 at National Public Health Laboratory (NPHL), Kathmandu, with an aim to assess the rate of S. aureus nasal carriage and MRSA carriage among HIV-infected and non-HIV patients. A total of 600 nonrepeated nasal swabs were analyzed following standard microbiological procedures, where 300 swabs were from HIV-infected patients while remaining 300 were from non-HIV patients. The isolates were identified on the basis of colony characteristics and a series of biochemical tests. The antibiotic susceptibility test (AST) was performed by the modified Kirby-Bauer disc diffusion method. Inducible clindamycin resistance in isolates was confirmed by the D-test method. RESULTS: Overall, out of 600 nasal swabs of patients tested, 125 (20.8%) were S. aureus nasal carriers which included 80 out of 300 (26.66%) among HIV-infected patients and 45 (15%) out of 300 among non-HIV patients, and the result was statistically significant (p=0.0043). Among the isolated S. aureus, 11 (13.8%) MRSA were confirmed in HIV-infected while 3 (6.7%) MRSA were detected from non-HIV patients. A higher number of S. aureus carriers was detected among HIV-infected males 40 (26.49%), whereas MRSA carriage was more prevalent among HIV-infected females 7 (5.1%). Among the HIV-infected, patients of age group 31-40 years were the ones with highest carriage rate 36 (45%), while in non-HIV patients, the highest rate 13 (28.9%) of carriage was detected among the patients of age group 21-30 years. Statistically significant difference was found between S. aureus carriage and HIV infection in patients (p < 0.05). Higher rate 2/3 (66.7%) of inducible clindamycin resistance in MRSA was detected from non-HIV patients in comparison to HIV-infected patients 7/11 (63.63%) while the result was statistically insignificant (p > 0.05). All the MRSA isolates (100%) were resistant against co-trimoxazole while ciprofloxacin showed high rate of sensitivity towards both MSSA and MRSA. None of the isolates were detected as VRSA. The major factors associated with nasal colonization of S. aureus were close personal contact, current smoking habit, and working or living in a farm (p < 0.05). CONCLUSION: Regular surveillance and monitoring of MRSA nasal carriage and antibiotic susceptibility pattern are of prime importance in controlling S. aureus infections especially in high risk groups like HIV-infected patients.

Serotyping and antibiotic susceptibility patterns of Vibrio and Shigella isolates from diarrheal patients visiting a Tropical and Infectious Diseases Hospital in central Nepal.

BACKGROUND: Diarrheal diseases are the major infectious disease in developing countries like Nepal. Lack of proper sanitation and antimicrobial resistance gained by microbes have challenged to address diarrheal diseases in resource-limited countries. Early diagnosis of disease and proper antibiotic treatment can significantly reduce the disease burden. This study was designed to determine the recent antimicrobial susceptibility pattern of Vibrio cholerae and Shigella spp. to assure the proper antibiotic treatment. Stool specimens were processed following microbiological protocol and identified by biochemical and serological tests recommended by the Clinical Laboratory Standard Institute. RESULTS: Out of total 640 analyzed stool samples, 50 were culture positive, among them 29 were Shigella spp. (64.4%) and 21 were V. cholerae (46.6%). All V. cholerae strains belonged to the serogroup O1 and serovar Ogawa. Among the Shigella spp., Shigella flexneri 17 (59%) topped the list of serotype followed by Shigella sonnei 8 (28%), Shigella dysenteriae 3 (10%) and Shigella boydii 1 (3%) respectively. All the V. cholerae isolates (100%) were sensitive to cefotaxime while 71% were sensitive to tetracycline but 100 and 90.4% were resistance to co-trimoxazole and nalidixic acid respectively. Shigella isolates were mostly susceptible to cefotaxime (97%) while ciprofloxacin (48%) and ofloxacin (55%) were less effective drugs. CONCLUSIONS: These results on the prevalence of enteropathogens and their antibiotic resistance pattern may help to guide accurate choice of therapy in clinical setting. Hence, development of evidence based National Guidelines for the treatment of diarrhea is needed.

Shigellosis in Nepal: 13 years review of nationwide surveillance.

BACKGROUND: Shigella is a major cause of gastroenteritis especially in children. In developing countries, the incidence is frequent and results are often life threatening. Changing epidemiology and emerging antibiotic resistance warrants continuous monitoring of susceptibility. The present study highlights the changing epidemiology and drug resistance patterns of Shigella isolated at different hospitals of Nepal over a period of 13 years (Jan. 2003-Dec. 2015). METHODS: This study was carried out in 12 participating laboratories. Stool specimens received at respective laboratories were processed for isolation and identification of Shigella species and confirmed by serotyping at National Public Health Laboratory. Antimicrobial resistance patterns were determined by Kirby Baeur disc diffusion test. RESULTS: A total of 332 isolates were identified as Shigella species of which Shigella flexneri (50 %) was the predominant serotype. Shigella dysenteriae, Shigella sonnei, Shigella boydii, and untypable Shigella spp. respectively, accounted for 28.6, 27.54, 10.2, 4.5, and 6.6 % of the total number. Change in prevalent serotype is noted over the years. S. dysenteriae was the prevalent species in Nepal in 2003 and 2004, but since 2005, S. flexneri remained prevalent. Majority of the isolates were recovered from children aged 1-10 years and was statistically significant (p = 0.023) compared to the other age groups. High resistance among all Shigella species to the first-line drugs like ampicillin (88 %), cotrimoxazole (76 %), ciprofloxacin (39 %,) and nalidixic acid (80 %) was observed; 46.1 % of total isolates were multidrug resistant (MDR), and the most common MDR profile was ampicillin, nalidixic acid, and co-trimoxazole. Prevalence of MDR increased significantly in 2010 as compared to 2003. Only few Shigella isolates were resistant to ceftriaxone. CONCLUSIONS: The study revealed S. flexneri as the predominant serogroup in Nepal. Children below 10 years were more prone to the disease. Nalidixic acid, ampicillin, co-trimoxazole, and ciprofloxacin should not be used empirically as the first-line drugs in treatment of shigellosis. Since the distribution of different species of Shigella and their antibiotic susceptibility profile may vary from one geographical location to another and may also change with time, continuous local monitoring of resistance patterns is necessary for appropriate antimicrobial therapy.

Design, construction and validation of a Plasmodium vivax microarray for the transcriptome profiling of clinical isolates.

High density oligonucleotide microarrays have been used on Plasmodium vivax field isolates to estimate whole genome expression. However, no microarray platform has been experimentally optimized for studying the transcriptome of field isolates. In the present study, we adopted both bioinformatics and experimental testing approaches to select best optimized probes suitable for detecting parasite transcripts from field samples and included them in designing a custom 15K P. vivax microarray. This microarray has long oligonucleotide probes (60mer) that were in-situ synthesized onto glass slides using Agilent SurePrint technology and has been developed into an 8X15K format (8 identical arrays on a single slide). Probes in this array were experimentally validated and represents 4180 P. vivax genes in sense orientation, of which 1219 genes have also probes in antisense orientation. Validation of the 15K array by using field samples (n=14) has shown 99% of parasite transcript detection from any of the samples. Correlation analysis between duplicate probes (n=85) present in the arrays showed perfect correlation (r2=0.98) indicating the reproducibility. Multiple probes representing the same gene exhibited similar kind of expression pattern across the samples (positive correlation, r≥0.6). Comparison of hybridization data with the previous studies and quantitative real-time PCR experiments were performed to highlight the microarray validation procedure. This array is unique in its design, and results indicate that the array is sensitive and reproducible. Hence, this microarray could be a valuable functional genomics tool to generate reliable expression data from P. vivax field isolates.

A cross strain Plasmodium falciparum microarray optimized for the transcriptome analysis of Plasmodium falciparum patient derived isolates.

Malarial parasite P. falciparum, an apicomplexan protozoan has a 23.3 MB nuclear genome and encodes ~ 5600 transcripts. The genetic diversity of the parasite within and across geographical zones is a challenge to gene expression studies which are essential for understanding of disease process, outcome and developing markers for diagnostics and prognostics. Here, we describe the strategy involved in designing a custom P. falciparum 15K array using the Agilent platform and Genotypic's Right Design methodology to study the transcriptome of Indian field isolates for which genome sequence information is limited. The array contains probes representing genome sequences of two distinct geographical isolates (i.e. 3D7 and HB3) and sub-telomeric var gene sequences of a third isolate (IT4) known to adhere in culture condition. Probes in the array have been selected based on their efficiency to detect transcripts through a 244K array experimentation. Array performance for the 15K array, was evaluated and validated using RNA materials from P. falciparum clinical isolates. A large percentage (91%) of the represented transcripts was detected from Indian P. falciparum patient isolates. Replicated probes and multiple probes representing the same gene showed perfect correlation between them suggesting good probe performance. Additional transcripts could be detected due to inclusion of unique probes representing HB3 strain transcripts. Variant surface antigen (VSA) transcripts were detected by optimized probes representing the VSA genes of three geographically distinct strains. The 15K cross strain P. falciparum array has shown good efficiency in detecting transcripts from P. falciparum parasite samples isolated from patients. The low parasite loads and presence of host RNA makes arrays a preferred platform for gene expression studies over RNA-Seq.

Disease specific modules and hub genes for intervention strategies: A co-expression network based approach for Plasmodium falciparum clinical isolates.

Systems biology approaches that are based on gene expression and bioinformatics analysis have been successful in predicting the functions of many genes in Plasmodium falciparum, a protozoan parasite responsible for most of the deaths due to malaria. However, approaches that can provide information about the biological processes that are active in this parasite in vivo during complicated malaria conditions have been scarcely deployed. Here we report the analysis of a weighted gene co-expression based network for P. falciparum, from non-cerebral clinical complications. Gene expression profiles of 20 P. falciparum clinical isolates were utilized to construct the same. A total of 20 highly interacting modules were identified post network creation. In 12 of these modules, at least 10% of the member genes, were found to be differentially regulated in parasites from patient isolates showing complications, when compared with those from patients with uncomplicated disease. Enrichment analysis helped identify biological processes like oxidation-reduction, electron transport chain, protein synthesis, ubiquitin dependent catabolic processes, RNA binding and purine nucleotide metabolic processes as associated with these modules. Additionally, for each module, highly connected hub genes were identified. Detailed functional analysis of many of these, which have known annotated functions underline their importance in parasite development and survival. This suggests, that other hub genes with unknown functions may also be playing crucial roles in parasite biology, and, are potential candidates for intervention strategies.

Plasmodium falciparum complicated malaria: Modulation and connectivity between exportome and variant surface antigen gene families.

In temperate and sub-tropical regions of Asia and Latin America, complicated malaria manifested as hepatic dysfunction or renal dysfunction is seen in all age groups. There has been a concerted focus on understanding the patho-physiological and molecular basis of complicated malaria in children, much less is known about it in adults. We report here, the analysis of data from a custom, cross strain microarray (Agilent Platform) using material from adult patient samples, showing hepatic dysfunction or renal failure. These are the most common manifestations seen in adults along with cerebral malaria. The data has been analyzed with reference to variant surface antigens, encoded by the var, rifin and stevor gene families. The differential regulation profiles of key genes (comparison between Plasmodium falciparum complicated and uncomplicated isolates) have been observed. The exportome has been analyzed using similar parameters. Gene ontology term based functional enrichment of differentially regulated genes identified, up-regulated genes statistically enriched (P<0.05) to critical biological processes like generation of precursor metabolite and energy, chromosome organization and electron transport chain. Systems network based functional enrichment of overall differentially regulated genes yielded a similar result. We are reporting here, up-regulation of var group B and C genes whose proteins are predicted to interact with CD36 receptor in the host, the up-regulation of domain cassette 13 (DC13) containing var group A, as also the up-regulation of group A rifins and many of the stevors. This is contrary to most other reports from pediatric patients, with cerebral malaria where the up-regulation of mostly var A group genes have been seen. A protein-protein interaction based network has been created and analysis performed. This co-expression and text mining based network has shown overall connectivity between the variant surface antigens (VSA) and the exportome. The up-regulation of var group B and C genes encoding PfEMP1 with different domain architecture would be important for deciding strategies for disease prevention.