Lower respiratory tract infections are considered a common cause responsible for morbidity and mortality among children, and Mycoplasma pneumoniae is identified to be responsible for up to 40 per cent of community-acquired pneumonia in children greater than five years of age. Extrapulmonary manifestations have been reported either due to spread of infection or autoimmune mechanisms. Infection by M. pneumoniae has high incidence and clinical importance but is still an underrated disease. Most widely used serologic methods are enzyme immunoassays for detection of immunoglobulin M (IgM), IgG and IgA antibodies to M. pneumoniae, though other methods such as particle agglutination assays and immunofluorescence methods are also used. Detection of M. pneumoniae by nucleic acid amplification techniques provides fast, sensitive and specific results. Utilization of polymerase chain reaction (PCR) has improved the diagnosis of M. pneumoniae infections. Besides PCR, other alternative amplification techniques include (i) nucleic acid sequence-based amplification, (ii) Qβ replicase amplification, (iii) strand displacement amplification, (iv) transcription-mediated amplification, and (v) ligase chain reaction. Macrolides are used as the first-line treatment in childhood for M. pneumoniae infections; however, emergence of macrolide-resistant M. pneumoniae is a cause of concern. Development of a safe vaccine is important that gives protective immunity and would be a major step in reducing M. pneumoniae infections.

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Background & objectives: The treatment of unruptured intracranial aneurysms (IAs) remains controversial; the ability to predict the risk of rupture for an aneurysm would be of clinical value. The aim of this study was to determine and evaluate the predictive value of the risk factors of IA rupture. Methods: This retrospective study involved 379 consecutive patients with 441 aneurysms between August 2011 and July 2014. Based on clinical data and computed tomography angiography findings, the potential of risk factors to predict the aneurysmal rupture was assessed using statistical methods. Results: Age, hypertension, heart disease, diabetes mellitus, cerebral atherosclerosis, aneurysms located at the internal carotid artery (ICA) and neck width (N) correlated negatively with rupture risk. Aneurysms located at the anterior communicating artery, bifurcation, irregularity, with a daughter sac, aneurysm height, maximum size, aspect ratio (AR), height-to-width ratio and bottleneck factor were significantly and positively correlated with rupture risk. The multivariate logistic regression model revealed that bifurcation aneurysm, irregular aneurysm and high AR increased the rupture risk, while cerebral atherosclerosis, aneurysm located on the ICA and greater N decreased the risk. Receiver operating characteristic analysis of AR curve values differed according to circumstances. Interpretation & conclusions: Cerebral atherosclerosis, location in the ICA and larger N were the protective factors against aneurysm rupture, and IAs located at bifurcations, irregular shape and increased AR indicated a greater rupture risk.

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Background & objectives: There are reports about the susceptibility of Aedes mosquitoes to ZIKV from various countries, however, no such information is available from Indian sub-continent, although, high level of group cross-reactivity of ZIKV with other flaviviruses has been reported. During outbreak situations, many cases of Dengue (DEN) and Chikungunya (CHIK) are reported. In such scenario, vector mosquitoes are likely to get co-infection/secondary-infection with one or other virus. The present study was carried out to determine the susceptibility of Indian strain of Aedes aegypti to Zika virus (ZIKV) strain (MR-766) and the effect of co-infection/super-infection with either dengue virus (serotype-2) (DENV) or chikungunya virus (CHIKV) on ZIKV replication. Methods: Ae. aegypti mosquitoes used in this study were reared for many generations since 1980 at laboratory colony maintained at the ICMR-National Institute of Virology, Pune, India. Transmissibility of ZIKV from infected mosquitoes to suckling mice was also studied. Mosquitoes were experimentally infected with ZIKV and super-infected with either DENV or CHIKV via membrane-feeding route and incubated for 14 days at 28±2°C and humidity of 85±5 per cent. Replication of these viruses in mosquitoes was confirmed using real-time reverse transcription-polymerase chain reaction and immunofluorescence assay. Twenty infected mosquitoes were allowed to feed upon four suckling CD1 mice for about 30 min. Transmission of the ZIKV by infected mosquitoes to suckling mice was confirmed by the appearance of clinical signs and the presence of viral RNA in different organs. Results: Concomitant infection of mosquitoes with all the three viruses showed simultaneous propagation of all three viruses, confirmed by real time RT-PCR and IFA. Infection of mosquitoes with CHIKV followed by ZIKV showed positivity in individual head squashes (7%) for both viruses using IFA; only 8.3 per cent showed dual positivity with primary infection of ZIKV followed by DENV; 8.3 per cent dual infection positivity was observed when infected with DENV followed by ZIKV; 5 per cent showed dual infection was observed when infected with ZIKV followed by CHIKV. Ae. aegypti was found to be susceptible to ZIKV strain as ZIKV could be detected from the second post-infection day (PID) in infected mosquitoes. Transmission of ZIKV to mice by the bite of infected Ae. aegypti establishes this species as a potential vector. Interpretation & conclusions: From super-infection experiments, it was concluded that ZIKV might have a relative advantage in replication dynamics over DENV. Vertical transmission was not observed for ZIKV in experimentally infected mosquitoes (n=920 larvae). Further studies are required to understand the possibility of silently circulating ZIKV in India, which remain non-detected because of lack of surveillance.

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Considerable progress has been made in the field of leptospiral vaccines development since its first use as a killed vaccine in guinea pigs. Despite the fact that the immunity conferred is restricted to serovars with closely related lipopolysaccharide antigen, certain vaccines have remained useful, especially in endemic regions, for the protection of high-risk individuals. Other conventional vaccines such as the live-attenuated vaccine and lipopolysaccharide (LPS) vaccine have not gained popularity due to the reactive response that follows their administration and the lack of understanding of the pathogenesis of leptospirosis. With the recent breakthrough and availability of complete genome sequences of Leptospira, development of novel vaccine including recombinant protein vaccine using reverse vaccinology approaches has yielded encouraging results. However, factors hindering the development of effective leptospiral vaccines include variation in serovar distribution from region to region, establishment of renal carrier status following vaccination and determination of the dose and endpoint titres acceptable as definitive indicators of protective immunity. In this review, advancements and progress made in LPS-based vaccines, killed- and live-attenuated vaccines, recombinant peptide vaccines and DNA vaccines against leptospirosis are highlighted.

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Background & objectives: The determination of cystatin C (cysC) may be helpful in diagnosis and monitoring of cancer because the pathogenesis of cancer is linked with an increased activity of cysteine peptidases (cathepsins) and a decrease of cysC concentration. This study was aimed to examine the utility of cysC as a marker of bladder cancer (BCa) to be used in the diagnosis. Methods: This study was conducted with 90 patients with BCa and 27 healthy people. Patients with other cancers, inflammation process and impaired renal function were excluded from the study. The concentrations of cysC in the plasma and urine were measured by surface plasmon resonance imaging technique. Results: The concentration of cysC in the serum taken from the patients with BCa [0.35±0.02 μg/ml (range: 0.20-0.78 μg/ml)] was significantly (P<0.001) lower than the serum cysC concentration of the healthy people [0.68±0.05 μg/ml (range: 0.52-0.89 μg/ml)]. The urinary cysC concentration of the BCa patients [0.19±0.01 μg/ml (range: 0.09-0.34 μg/ml)] was not significantly different from the urinary cysC concentration of the healthy people [0.24±0.02 μg/ml (range: 0.16-0.33 μg/ml)]. Receiver operating characteristic (ROC) curve showed that BCa patients with cysC concentration <0.54 μg/ml [sensitivity: 87%; specificity: 92%; area under the curve (AUC) of ROC: 0.927; P=0.02] could be optimally separated from healthy people. The ROC curve further showed that superficial low-grade patients with cysC concentration lower than 0.36 μg/ml (sensitivity: 0.63%; specificity: 0.58%; AUC of ROC: 0.635; P=0.08) could not be optimally separated from high-risk tumour patients. Interpretation & conclusions: BCa patients have lower serum cysC concentration than the control group. Serum cysC may be considered as a potential marker of BCa but not its aggressiveness.

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Background & objectives: New Delhi metallo-β-lactamase 1 (NDM-1) cleaves the beta-lactam ring, and confers bacterial resistance against most of the beta-lactam antibiotics, except tigecycline and colistin. Among these two antibiotics, colistin is considered toxic, and therefore, its clinical use and dosage need cautious approach. In the present study, six organic acids were screened individually and in combination of two acids for their effectiveness against NDM-1 Escherichia coli and a combination of colistin and oxalic or succinic acid was tested to find out the potential of combination therapy for reducing the dose of toxic colistin. Methods: Antibacterial activity of the organic acid and their combinations was tested by disc diffusion method against NDM-1 E. coli, and minimum inhibitory concentration (MIC) was determined by broth dilution method. Synergistic effect between organic acids and colistin was tested by checkerboard method. Results: Oxalic acid showed the highest zone of inhibition (15±1 mm) followed by succinic acid, tartaric acid, fumaric acid, citric acid and malic acid. The combination of two acids did not increase the zone of inhibition significantly. MIC was found to be the lowest with oxalic acid and succinic acid (320 μg/ml). In the presence of 160 μg/ml oxalic acid or succinic acid, MIC of colistin was reduced from 8 to 4 μg/ml, indicating synergistic effect. Interpretation & conclusions: Our findings showed that combination therapy using colistin and oxalic acid or succinic acid might find safe clinical application of this antibiotic in controlling infections due to NDM-1 bacteria.

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Background & objectives: High prevalence of certain polymorphic alleles of erythrocytes in malaria endemic area has been linked to the resistance provided by these alleles against parasitic infestations. Numerous studies undertaken to demonstrate this correlation have generated conflicting results. This study was undertaken to investigate the abilities of various polymorphic erythrocytes to support in vitro growth of Plasmodium falciparum parasites. Methods: In this study under in vitro condition the ability of P. falciparum parasites to grow was assessed in the erythrocytes obtained from a total of 40 patients with various haemoglobinopathies, such as β-thalassaemia (β-Thal), sickle cell anaemia, erythroenzymopathy-like glucose-6-phosphate dehydrogenase deficiency and membranopathy-like hereditary spherocytosis. Results: Significantly reduced in vitro invasion and growth of parasites was seen in the cultures containing abnormal erythrocytes than in control cultures containing normal erythrocytes (P< 0.05). The mean per cent parasitaemia comparison was also carried out among the three polymorphic erythrocyte groups, i.e. β-Thal, sickle cell anaemia and enzyme-membranopathies. Interpretation & conclusions: Erythroenzymopathies and membranopathies were found to provide a more hostile environment for parasites, as the least parasitaemia was observed in these erythrocytes. The present in vitro study showed that P. falciparum did not grow well and did not invade well in erythrocytes obtained from common inherited red cell disorders.

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Background & objectives: Brucellosis can lead to haematological abnormalities including cytopenia confusing with haematological malignancies. The aim of this study was to compare the main characteristics of brucellosis patients without cytopenia (Group 1) and with cytopenia (Group 2). Methods: This five-year period study which was performed in two referral hospitals in Turkey, included all adult brucellosis patients. Abnormally, low counts of leucocyte or haemoglobin or platelets in a patient were considered as cytopenia. The demographics, clinical, laboratory, treatment and outcome data were analyzed. Results: A total of 484 brucellosis patients were enrolled. Among the cases, 162 (33.5%) of them had cytopenia. One hundred and four (21.5%) had anaemia, 88 (18.8%) had thrombocytopenia, 71 (14.6%) had leucopenia and 28 (5.8%) had pancytopenia. The mean age of group 2 was 35.01±16.05 yr and it was 33.31±14.39 yr in group 1. While there was no difference between the groups in terms of duration of treatment, the median length of hospital stay (LOS) was significantly longer in group 2 (9 vs 10 days; P<0.001). The most frequently applied combination therapy consisted of doxycycline plus rifampicin and doxycycline plus streptomycin regimens. No significant difference was observed in terms of duration of treatment, LOS and restoration time of cytopenia between the patients who received either of these combinations. Interpretation & conclusions: Our findings suggested that the patients with cytopenia should be investigated for brucellosis, especially if living in, or with a history of travel to, endemic areas, in view of the increase in world travel.

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Background & objectives: The incidence and severity of invasive and non-invasive infections demonstrate variability over time. The emerging resistance of Group A streptococci (GAS) to commonly used antibiotics is of grave concern. This study was conducted to assess the antimicrobial resistance of beta-haemolytic streptococci (βHS) in India and to ascertain the molecular mechanisms of resistance. Methods: All isolates of βHS from the Trauma Centre of All India Institute of Medical Sciences (AIIMS) (north India), and heavily populated area of old Delhi from 2010 to 2014 and Yashoda Hospital, Secunderabad (in south India, 2010-2012) and preserved isolates of βHS at AIIMS (2005-2009) were included. Phenotypic confirmation was done using conventional methods and the Vitek 2. Antibiotic sensitivity testing was done by disc diffusion and E-test. Detection of resistance genes, erm(A), erm(B), mef(A), tet(M) and tet(O), was done by polymerase chain reaction (PCR). Results: A total of 296 isolates of βHS (240 from north and 21 from south India) were included in the study. Of the 296 βHS, 220 (74%) were GAS, 52 (17.5%) were Group G streptococci and 11 (3.7%), 10 (3.3%) and three (1%) were Group B streptococci, Group C streptococci and Group F streptococci, respectively. A total of 102 (46%) and 174 (79%) isolates were resistant to tetracycline and erythromycin, respectively; a lower resistance to ciprofloxacin (21, 9.5%) was observed. A total of 42 (14%) and 30 (10%) isolates, respectively, were positive for tet(M) and erm(B) genes. Only 13 (5%) isolates were positive for mef(A). None of the isolates were positive for erm(A) and tet(O). There was discordance between the results of E-test and PCR for erythromycin and tetracycline. Interpretation & conclusions: A high level of resistance to erythromycin and tetracycline was seen in βHS in India. Discordance between genotypic and phenotypic results was reported. Absence of erm(A) and tet(O) with high prevalence of tet(M) and erm(B) was observed.

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Background & objectives: Though Unani medications have been used for centuries to treat psoriasis, there is paucity of published studies which have systematically evaluated their efficacy and safety. This study was conducted to establish non-inferiority of Unani medications (oral UNIM-401 and topical UNIM-403) vs psoralen plus ultraviolet A (PUVA) sol in treatment of moderate-severe chronic plaque psoriasis (CPP) in achieving psoriasis area severity index (PASI) 75 at 12 wk and to estimate proportion of patients who relapsed in follow up period of 12 weeks, after having achieved PASI 50. Methods: In this randomized, controlled trial patients with CPP were block randomized to receive either Unani treatment (147 patients) or PUVA sol (140 patients) for 12 weeks. Percentage reduction in PASI was determined in each patient at 12 wk to calculate number of patients who achieved PASI 75 as also to estimate median of percentage reduction in PASI in each group. All patients who achieved PASI 50 at 12 weeks were followed up for another 12 wk to determine proportion of patients who relapsed. Results: Of the 287 patients randomized, 84 of 147 in Unani group and 67 of 140 in PUVA sol group completed 12 weeks of treatment. On intention-to-treat (ITT) analysis, the response in patients on Unani medication was not inferior to those receiving PUVA sol, in attaining PASI 75 (16.3% in Unani group vs 15.7% in the PUVA sol group). Median of percentage reduction of PASI at 12 wk from baseline in Unani group (68.2%; −60, 100) and PUVA sol group (63%; −15.7, 100) was comparable. Proportion of patients who relapsed at 24 wk was comparable in both groups. However, frequency of clinical side effects was significantly higher (P =0.001) in PUVA sol group (16.4%) compared to Unani group (2%). Interpretation & conclusions: The findings of the present study indicated that oral UNIM-401 and topical UNIM-403 were effective and well tolerated therapeutic options in patients with moderate-severe CPP.

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Background & objectives: Biolarvicides may offer alternatives to chemical larvicides as these are known to be safe to environment and selective against the target species. However, only a limited number of biolarvicides have been approved for mosquito larval control. In the current study, a new formulation of spinosad, 20 per cent emulsifiable concentrate (EC) was tested for its efficacy against Culex quinquefasciatus, in comparison to its 12 per cent suspension concentrate (SC). Methods: Spinosad 20 per cent EC was tested against Cx. quinquefasciatus immature at 25, 50, 100 and 150 mg active ingredient (ai)/m^[2] in cesspits, drains and abandoned wells in comparison with spinosad 12 per cent SC at the optimum field application dosage of 50 mg ai/m^[2]. Results: The 20 per cent EC caused 90-100 per cent reduction of pupal density for 7-14 days in cesspits, 10-17 days in drains and 14-30 days in abandoned wells at all dosages tested. At lower dosages of 25 and 50 mg ai/m^[2], >90 per cent reduction of pupal density was observed for one week in cesspits and street drains and for two weeks in abandoned wells. The effective duration of control provided by the higher dosages, 100 and 150 mg ai/m^[2] was 1.4 to 2 times greater than the lower dosages, 25 and 50 mg ai/m^[2]. Interpretation & conclusions: The findings showed that the spinosad 20 per cent EC can be used for larval control against Cx. quinquefasciatus, at the dosage of 25 mg ai/m^[2] at weekly interval in cesspits and drains and at fortnightly interval in abandoned wells. Spinosad 20 per cent EC could be one of the options to be considered for larval control under integrated vector management.

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Background & objectives: Despite advancements in molecular-based methods, the composition of the human ileal microbiota and the effects of synbiotics/probiotics on its microbes remain poorly understood. The aim of this study was to determine the composition of the mucus microbiota in the human ileum and to assess the effects of oral administration of synbiotics on the microbiota. Methods: As part of a clinical trial for synbiotics treatment and surgical infection, ileal mucus was sampled when resection of the ileocecal portion was required. The microbiota composition was examined using 16S rRNA-targeted real-time-quantitative polymerase chain reaction. Results: A total of 33 samples from the synbiotics group and 39 from the control group were analyzed. Total numbers of bacteria in the ileum were 10^8.5 cells/g in the synbiotics group and 10^8.4 cells/g in the control group, in which obligate anaerobes were dominant over facultative anaerobes. The level of Enterobacteriaceae was significantly lower in the synbiotics group than in the control group. The administered probiotics species Lactobacillus casei strain Shirota and Bifidobacterium breve strain Yakult were detected in 42 and 76 per cent of the synbiotics group, respectively. No significant correlations were observed between tumour stage/size and the various microbes present, except for a negative correlation between tumour size and Bifidobacterium. Interpretation & conclusions: The present analysis of a substantial number of samples from surgically resected intestines showed an abundance of obligate anaerobes as a characteristic feature of the ileal mucus microbiota. Our results also indicated that the synbiotics intervention induced a prominent reduction in Enterobacteriaceae in the ileal microbiota.

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Background & objectives: Ocular and adnexal tumours are important causes of morbidity in India and globally. Immunohistochemistry (IHC) is a vital molecular pathology tool, which helps to diagnose a tumour with more accuracy. The present study was undertaken to document the profile of ocular and adnexal tumour with IHC at a tertiary eye care center in Northeast India. Methods: This was a prospective and laboratory-based study. Histopathological and IHC study of the ocular and adnexal tumour was carried out from 2012 to 2014. Selection of pathological cases was made on the result of the histological diagnosis. All samples were subjected to IHC using kits for different antibodies as per indications. Results: In total, 645 tumours were included in our study, with 449 benign conditions and 196 were malignant tumours. Total IHCs were done in 87 tumours and 238 of antibodies were used. Non-Hodgkin's lymphomas (B-cell, low-to-intermediate type and mucosal-associated lymphoid tumours) were the most common tumor. Interpretation & conclusions: Clinical utility of the IHCs in different ophthalmic tumours can enable pathologists to make an accurate diagnosis and thus help in the overall management of the patient care. IHC may be carried out using various methods and some of the methods practiced are time consuming and tedious. In this study, kit methods were used which were found to be simpler and less time-consuming.

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