Diagnosis of gonorrhoea is an ongoing challenge. The organism is fastidious requiring meticulous collection and transport for successful cultivation. Asymptomatic infections are common which go undetected by conventional methods thereby leading to continued transmission and the risk of complications. The nucleic acid amplification tests, now increasingly used in developed countries, offer improved sensitivity compared to bacterial culture. However, these continue to suffer sequence related problems leading to false positive and false negative results. Further, these cannot be used for generation of data on antibiotic susceptibility because genetic markers of antibiotic resistance to recommended therapies have not been fully characterised. They are unaffordable in a setting like ours where reliance is placed on syndromic approach for sexually transmitted infection (STI) management. The use of syndromic approach has resulted in a considerable decline in the number of Neisseria gonorrhoeae isolates that have been cultured for diagnostic purposes. Many laboratories formerly doing so are no longer performing culture for gonococci, and the basic skills have been lost. There is a need to not only revive this skill but also adopt newer technologies that can aid in accurate diagnosis in a cost-effective manner. There is room for innovation that can facilitate the development of a point-of-care test for this bacterial STI.

Objectives: Both cysteine proteinase inhibitors (CPIs) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) play important roles in the pathogenesis of parasites and their relationship with the hosts. We constructed a new eukaryotic recombinant expression plasmid pcDNA3.1(+)-BmCPI/BmGAPDH of periodic Brugia malayi for investigation of the DNA vaccine-elicited immune responses. Materials and Methods: We cloned a gene encoding the CPIs and GAPDH from periodic B. malayi into vector pcDNA3.1. The composited plasmid or the control was injected into the tibialis anterior muscle of the hind leg in BALB/c mice, respectively. The target genes were detected by reverse transcription-polymerase chain reaction in muscle tissues. The stimulation index (SI) of T-lymphocyte proliferation and the levels of interferon-gamma (INF-g) and interleukin-4 ( IL-4) in serum were detected by thiazolyl blue tetrazolium blue and enzyme-linked immunosorbent assays. Results: The pcDNA3.1(+)-BmCPI/BmGAPDH was amplified from muscle tissues of the mice after immunisation. The SI of the immunised group was significantly higher than that of the two control groups (P < 0.05). The levels of INF-g and IL-4 of pcDNA3.1(+)-BmCPI/BmGAPDH group were both higher than those of the two control groups (P < 0.05). The level of INF-g of pcDNA3.1(+)-BmCPI/BmGAPDH group was significantly higher than that of pcDNA3.1(+)-BmCPI/CpG group (P < 0.05). Conclusions: We conclude that the recombinant plasmid pcDNA3.1(+)-BmCPI/BmGAPDH could elicit specific humoural and cellular immune responses in mice.

Purpose: To describe the microbiological profile and clinical outcome in the eyes with culture-proven exogenous endophthalmitis. Methods: A retrospective analysis of 495 eyes diagnosed as exogenous endophthalmitis was performed over a period of 10 years. In all, aseptically collected aqueous and vitreous aspirates were cultured for bacteria and fungus using standard microbiological techniques. Gram-stain and KOH preparation of the specimens were also performed. The antibiotic susceptibility testing for bacterial isolates was performed by Kirby-Bauer disk diffusion method. The treatment was modified according to the antibiotic sensitivity profile. The final clinical ocular condition was divided into improved, stable or deteriorated. Results: Of 148 culture-proven endophthalmitis eyes, 137 (92.57%) were referred from elsewhere, and 11 (7.43%) belonged to our institute. Aetiologically, 76 (51.35%) eyes were post-cataract surgery, 61 (41.22%) were post-traumatic, 5 (3.38%) eyes post-intravitreal anti-vascular endothelial growth factor injection, 5 associated with corneal diseases and 1 bleb-related endophthalmitis. In 31 (20.95%) eyes, primary intravitreal antibiotics were given outside. The cultures revealed monomicrobial growth in 92.57% (n = 137) and polymicrobial growth in 7.43% (n = 11). Among the bacteria (n = 121, 81.76%), Pseudomonas species dominated overall (n = 32, 27.11%) and post-operative (n = 26, 38.23%) endophthalmitis group. Staphylococcus epidermidis (n = 14, 28%) was prominent in post-traumatic endophthalmitis group. Ninety-two percent (n = 108 isolates) of bacteria were sensitive to vancomycin. In 78 (52.7%) eyes, the clinical ocular condition improved or remained stable while deteriorated in 51 (34.46%). Conclusion: A bacterial predominance was observed among causative organisms of exogenous endophthalmitis with Pseudomonas species being the most common. The appropriate surgical intervention improved or stabilised the visual acuity in nearly 50% eyes.

Aims: Streptococcus pneumoniae, an opportunistic pathogen commonly carried asymptomatically in the nasopharynx of children, is associated with increasing rates of treatment failures due to a worldwide increase in drug resistance. We investigated the carriage of S. pneumoniae in children 5 years or younger, the identity of prevalent serotypes, the rates of resistance to macrolides and other antimicrobial agents and the genotypes responsible for macrolide resistance. Materials and Methods: Nasopharyngeal swabs were collected from 157 children under 5 years for cultural isolation of S. pneumoniae. Antibiogram of isolates  was determined using the disk diffusion test, and the minimal inhibitory concentration to macrolides was determined using the E-test. Isolate serotypes and macrolide resistance genes, erm(B) and mef(E), were identified using multiplex polymerase chain reactions. Results: S. pneumoniae was recovered from 33.8% of children; 41.9% among males and 21.9% among females (P = 0.009). The highest carriage rate occurred among age groups 7-12 months and 49-60 months. Most frequent serotypes were 19F, 6A/B, 11A, 19A, 14 and 15B/C.  Resistance to macrolides was 60.4%. Resistance to oxacillin, trimethoprim/sulfamethoxazole and clindamycin was present among 90.6%, 54.7% and 32.1% of isolates, respectively. All isolates were susceptible to chloramphenicol, levofloxacin and vancomycin. Isolates resistant to one or more macrolide drugs were more likely to be multidrug resistant. Resistance to clindamycin or oxacillin coexisted with macrolide resistance. Among the erythromycin-resistant isolates, erm(B), mef(E) and erm(B) and mef(E) genes were present at rates of 43.8%, 37.5% and 6.3%, respectively. Erm(B) and mef(E) were associated with very high level and moderate-to-high level resistance to macrolides, respectively. Conclusion: A significant proportion of children harboured macrolide and multidrug-resistant S. pneumoniae.

Background: Severe acute hepatitis B (SAHB) is an insufficiently described clinical entity, with relatively scarce data on anti-viral therapy available in field literature. Methods: We performed an open-label study to evaluate specific anti-viral therapy in SAHB in Bucharest, Romania, during 2005-2009. Patients were allocated to two treatment groups and one control group: Group 1 - lamivudine 100 mg/day, Group 2 - entecavir 0.5 mg/day and Group 3 - standard of care, without anti-viral therapy. The primary endpoint was hepatitis B surface antigen (HBsAg) to hepatitis B surface antibody (anti-HBs) seroconversion by 24 weeks. Additional analyses included assessment of HBsAg clearance and hepatitis B e antigen (HBeAg) to hepatitis B e antibody (anti-HBe) seroconversion. Results: In Group 1, 7/69 patients (10.14%, P = 0.032) reached HBsAg/Ab seroconversion by 24 weeks, compared with 9/21 (42.85%, P = 0.053) in Group 2 and 25/110 (22.72%) in Group 3. HBsAg clearance by 24 weeks: 16/69 patients (23.18%, P = 0.027) in Group 1, 11/21 (52.38%, P = 0.256) in Group 2 and 43/110 (39.09%) in Group 3. HBeAg/Ab seroconversion: 46/61 (75.40%, P = 0.399) in Group 1, 9/19 (47.36%, P = 0.001) in Group 2 and 74/100 (74.00%) in Group 3. Conclusion: Anti-viral therapy can be considered for managing selected cases of SAHB. Biochemical as well as virological parameters need to orient the choice of the anti-viral agent. Lamivudine displayed a greater decrease in viral load compared to controls, but it was associated with lower levels of HBsAg to anti-HBs seroconversion. Patients treated with entecavir showed a better response in terms of HBs seroconversion by 24 weeks.

Background: The ability of microorganisms to evade antibiotic pressure is challenging in healthcare as patients have little or no drug treatment options. Detection of the prevalence of antibacterial resistance pattern helps towards improved antibiotic policy and empirical treatment. Objectives: We carried out antibiogram profiling and documented the prevalence and co-prevalence of New Delhi metallo-β-lactamase (NDM) and extended spectrum β-lactamases (ESBL) encoding genes in urinary Escherichia coli and Klebsiella pneumonia isolates. Materials and Methods: Antibiotic susceptibilities were tested for 241 isolates of E. coli and K. pneumoniae from urine samples collected from out- and hospitalised patients. Polymerase chain reaction (PCR) was carried out on isolates tested positive for phenotypic production of metallo-β-lactamase and ESBL. A multiplex PCR assay was designed to detect the genes. Results: Multiplex PCR assay designed had a limit of detection of 10 ³ CFU/mL in vitro. NDM detected was significantly higher among K. pneumoniae compared to E. coli (69.2% vs. 18.2%; P = 0.001). Of 17, 14 NDM positive isolates also harboured ESBL genes. The co-production of CTX-M + TEM + NDM (3/9; 33.3% and 5/8; 62.5%) was most common in K. pneumoniae and E. coli, respectively while CTX-M + TEM + SHV + NDM was found in one isolate. Of the 156 phenotypically ESBL producing isolates, CTX-M, TEM and SHV was detected by PCR in 85, 53 and 24 isolates, respectively. Conclusion: NDM and ESBL co-producing isolates were both community (64.7%) and hospital (35.29%) acquired among E. coli. Antibiotic resistance can be effectively evaluated by a cost and time effective molecular method, such as the multiplex PCR used in this study, which complement culture and sensitivity tests.

Introduction: Human Immunodeficiency Virus (HIV), Hepatitis B virus (HBV) and Hepatitis C virus (HCV) are the most common occupationally acquired infections amongst the healthcare workers (HCWs) with critically ill patients attending the emergency department being the most common source of occupationally acquired infections. Therefore, the present study was conducted at a 165 bedded level-1 trauma centre of India to ascertain the seroprevalence rate of HIV, HBV and HCV; and thus the risk associated with the occupational exposure in a busy emergency setting of the developing world. Methodology: A retrospective, 7 years study (2007-2013) was carried out at the JPNA Trauma Centre of the 2,500 bedded All India Institute of Medical Sciences, New Delhi. Records of all patients whose serum samples were sent to the laboratory for viral markers testing were obtained and those falling in the red area were included. Results: A total of 11,630 patients were received in the red area; and samples from 7,650 patients were sent for testing. Seropositivity of HIV, HBV and HCV in these samples was 0.28%, 3.4% and 0.9% respectively. The number of samples received was lesser than the total number of patients received in the red area. Conclusion: Adopting Standard Precautions (SP) can be used as an easy method to decrease the risk of occupationally acquired infections.

Purpose: The aim of this study was to isolate a novel mycobacteriophage and then explore its anti-tuberculosis (TB) potential. Materials and Methods: Phage was isolated from enriched soil sample. A total of 36 mycobacterial strains obtained from clinical specimens were subjected to investigate the host range of phage by the spot lysis assay. Biological characteristics were investigated through growth curve, host range and phage antimicrobial activity in vitro. Then, genome sequencing and further analysis were accomplished by using an ABI3730XL DNA sequencer and comparative genome, respectively. Results: A lytic mycobacteriophage (Chy1) was isolated and the plaque morphology was similar to D29. The genome of Chy1 was estimated to be about 47,198 base pair (bp) and strong similarity (97.4% identity) to D29, especially, the Chy1 gene 7 encoding holin which is considered as a clock controlling growth cycle of the corresponding phage, was identical (100% identity) to phage D29 gene 11, thus classifying Chy1 as a member of the cluster A2 family. However, to our surprise, Chy1 can infect a narrower range of host-mycobacterial strains than that of D29. The latent period of Chy1 was quite longer compared to D29. Moreover, Chy1 has a weaker ability to lyse Mycobacterium smegmatis compared to D29. Conclusions: The sequence of Chy1 showed 97.4% homology with the genome sequence of D29, but there was a large difference in their biological characteristics. Overall, the results of this investigation indicate that Chy1 is not an ideal candidate for developing mycobacteriophage-based anti-TB therapies but for future researches to investigate the reason why biological characteristics of Chy1 and D29 were remarkably different.

Objective: The present study aims to find out the mutational prevalence of Plasmodium falciparum chloroquine resistance transporter (Pfcrt) gene in Assam and Arunachal Pradesh, India. Methods: To fulfil the objective of the study, a total of 54 P. falciparum malaria positive samples were attempted for genotyping of Pfcrt gene using polymerase chain reaction (PCR) and direct DNA sequencing method. Results: The K76T mutation was observed in 77.78% cases followed by M74I (61.11%), N75E (61.11%) and C72S (16.67%). Triple mutant allele M74I+N75E+K76T was found in 61.11% P. falciparum field isolates. Double mutant allele C72S+K76T was seen among 16.67% samples. Mutation studies have shown existence of three different haplotypes of which two were associated with chloroquine resistance. The haplotype CVIET was found preponderance and circulated in all four districts. The other haplotype SVMNT was observed only in Karbi Anglong district of Assam. Maximum haplotype diversity was also recorded from Karbi Anglong district of Assam. Conclusion: Our study has confirmed high prevalence of mutant Pfcrt genotypes associated with chloroquine resistance in Assam and Arunachal Pradesh, India.

Background and Aim: To study the rate of wound infections in the post-discharged patient population and to assess the usefulness of post-discharge surveillance. Methods: A prospective surveillance of all the post-discharged trauma patients was done during a period of 6 months. Discharge instructions were given to all the patients regarding identification of the signs and symptoms of wound infections. They were telephonically followed up after a week to enquire about the wound condition and followed up in the outpatient department (OPD). Microbiology culture samples of those showing any signs and symptoms of infections were sent and their antimicrobial therapy, any change in the treatment schedule and the length of their hospital stay were noted. Factors such as wound class, type of surgeries and readmissions were noted. Results: A total of 281 postdischarge patients were enrolled, of which 101 were completely followed up for wound infections. Males were predominant (89%). Of the 101 patients, 42 (41.6%) patients wound showed infection during the intense follow-up in the OPD. However, 59 patients (59/101, 58.4%) showed wound swab culture positivity before discharge. These 42 patients developed signs and symptoms of infection post-discharge; 23 (22.7%) of them had change of antibiotic therapy during the follow-up period due to culture positivity. Acinetobacter sp., Staphylococcus aureus and Klebsiella pneumoniae were the predominant organisms isolated in the study. A total of 45 patients (44.5%) had to be readmitted due to wound site infections. Conclusions: Wound infections are common after discharge among trauma patients highlighting the importance of active surveillance and participation of patients.

Background: The most frequently isolated fungi in patients using TPN belongs to the Candida genus. Various infections including venous catheter infections, fungemia, endocarditis and ophthalmitis may be encountered. Objective: Upon growth of Candida in the blood cultures from the pediatric (neonatal) unit of our hospital, a surveillance was performed in this unit and involving the health care workers. Clonal relationships of the isolates were investigated with molecular tests. Methods: Blood samples obtained from the patients in pediatric neonatal unit were studied with automatized blood culture [BacT/Alert (Bio Mιrioux, France)]. Yeast isolates from environmental surveillance cultures (TPN solutions, hands of healthcare personnel, ιtagθre, etc) and patients were identified as C. albicans with conventional methods and ID 32 C and ATB ^TM Fungus 3 (Biomerieux, France) kits. Clonal similarity was determined by using AP-PCR as initial method and we have also typified all strains by the method of REP-PCR (diversilab system,bioMιrieux). Finally; Pulsed Field Gel Electrophoresis (PFGE) was used for confirmation. Results: C. albicans was isolated in blood cultures of seven patients. Similar antifungal susceptibility patterns were observed in all isolates. AP-PCR and REP-PCR showed that the C. albicans isolates grown in the TPN solution and from the patients' blood cultures were clonally same strains. PFGE analysis further confirmed this clonality. Conclusion: According to results of the molecular methods, we thought that a C. albicans outbreak had occurred in the neonatal pediatric unit, due to contamination of TPN solution.

Objective: To highlight the issue of freely available fixed-dose combinations (FDCs) of antimicrobials. Methods: A critique of two such antimicrobial FDCs was undertaken wherein the following aspects were assessed - rational and regulatory issues and justification for clinical use. Available in vitro, in vivo (animals and humans) evidence from published literature was analysed. Conclusions: There are several inadequately addressed aspects of the considered FDCs which are available in Indian market. In view of the growing problem of antimicrobial resistance, this issue must get the required attention.

A panel of 129 Giemsa-stained thick blood spots (TBS) confirmed for Plasmodium falciparum infection having different levels of parasite density were collected from a malaria endemic area. DNA was extracted and nested polymerase chain reaction (PCR) assay was performed to amplify P. falciparum DNA. Nested PCR assay successfully amplified P. falciparum DNA at a very low parasitaemia of ~10 parasites/μl of blood. Current PCR assay is very simple and can be used retrospectively to monitor the invasion and prevalence of different Plasmodium species in endemic areas.

Background: Chikungunya virus has recently re-emerged in India. Objectives: Assess prevalence of Chikungunya. Materials and Methods: Study conducted from April 2011 to September 2011. Two hundred and six patients (206) of both sexes (100 males and 106 females) of all age groups studied. Serum separated and CHIKV MAC IgM ELISA and Hemagglutination inhibition assay done. Results: 76 cases (36.89%) sero-positive by both the methods. Conclusion: Re-emergence and resurgence of the Chikungunya virus requires continuous monitoring.

To prevent the spread of carbapenemases-producing Enterobacteriaceae (CPE) active surveillance, contact isolation and cohorting infected patients should be practiced. Rectal swabs for the Xpert MDRO-assay of 32 patients were included. 71.85% were positive for targets incorporated into the MDRO-assay; whereas 28% were phenotypically not CRE and Xpert negative (9.37% had different mechanism [bla _OXA]). The assay identified 59.3%, 9.37% and 3.1% as bla _NDM, bla _NDM+VIM and bla _VIM, respectively. The assay is a screening test that identifies CPE harbouring organism within an hour and can be installed at tertiary-care facilities to screen colonized patients.

Early diagnosis and treatment of sepsis by appropriate antibiotics is of utmost importance. Therefore, we evaluated 16S rRNA panbacterial polymerase chain reaction (PCR) for rapid diagnosis of sepsis in 49 adult patients in Intensive Care Units (ICUs) and compared it with an automated blood culture. 8 ml of 10 ml blood collected was inoculated into BACTEC^® aerobic bottle and the remaining 2 ml was used for DNA extraction and PCR. 109 of 115 (93%) episodes of suspected sepsis showed concordant results between automated culture and PCR. Six episodes were positive by PCR only. Panbacterial PCR reduces turnaround time with rapid differentiation between systemic inflammatory response syndrome and sepsis.

Enteric fever is a public health problem with the upsurge in the occurrence of Salmonella isolates that are resistant to ciprofloxacin. In this study, a total of 284 blood culture isolates of S. Paratyphi A were investigated. Of these isolates, 281 (98.9%) were nalidixic acid resistant. A high rate (6.3%) of high-level resistance (≥4 μg/mL) was found to ciprofloxacin. The isolates with ciprofloxacin minimum inhibitory concentrations (MICs) of ≥12 μg/mL had 4 mutations, 2 mutations within the quinolone resistance-determining region of gyrA and 2 mutations also in parC. According to the Clinical Laboratory Standards Institute 2012 MIC breakpoints, 75.0% of isolates were resistant to ciprofloxacin. Finally, 3 major pulsed-field gel electrophoresis patterns were observed among the S. Paratyphi A isolates. The spread of fluoroquinolone resistant S. Paratyphi A necessitates a change toward 'evidence-based' treatment for enteric fever. The research provides a perspective on the increasing prevalence of antimicrobial resistant S. Paratyphi A isolates in this region of India.

The surveillance in Chennai identified 134 children and 443 adults clinically suspected for leptospirosis. Of these, 35 (26.1%) children and 118 (26.6%) adults had laboratory confirmed diagnosis for leptospirosis. The paediatric leptospirosis exhibited a higher frequency of classic features of Weil's disease. The prevalent serovar encountered was Icterohaemorrhagiae with no difference in the pattern of infecting serovars between the two groups. Further, confirmation of diagnosis was achieved by polymerase chain reaction (PCR) with a positivity of 28.4% (specificity 96%). Univariate analysis showed significant association of paediatric leptospirosis with rat infestation (odds ratio 87.4). Thus, PCR facilitates early diagnosis of febrile illness among paediatric cases.

We report an outbreak of acute viral hepatitis among children in a flood rescue camp at Rudraprayag district of Uttarakhand State, India. In May 2013, there was a disastrous natural calamity, The Himalayan Tsunami in Himalayan and Sub-Himalayan region of Uttarakhand. More than 5700 people were feared dead, and thousands were sheltered in different rescue camps. A linkage was hypothesised between the infected individuals and the common water sources feared of being contaminated faecally. Aetiological agent of the present outbreak was HAV that is emerging in an outbreak form in India, emphasizing a definite need for formulating mandatory vaccination and proper control strategies. The report exemplifies the basic problems encountered after a natural calamity.

Context: HCWs all over the world carry occupational risk of getting infected with major blood borne infections through needle stick injuries (NSIs). As health care industry has been expanding, risk of nosocomial infections is increasing proportionately. Measures to prevent it and put in place a mechanism to control these injuries are needed urgently, especially in India where there is not only increase in domestic demand but impetus in health tourism. Aim: To determine HBs Ag, HBc IgM level and to assess anti-HBs level prevalence in HCWs, in a tertiary care hospital and to study the influence of factors like age and sex in the vaccinated HCWs and formulate mechanism to increase awareness to create a safe working environment in the hospitals. Settings and Design: 437 HCWs, working in Laboratories, Surgical, Medical or Dental departments in 11 Civil Hospitals and Sub-district Hospitals covering 8 circles of the State. Methods and Material: Qualitative and Quantitative estimation of HBs Ag and Anti-HBs by sandwich ELISA technique and qualitative HBc IgM level by antibody-capture, non-competitive test. Liver profile (SGPT, SGOT and Alkaline Phosphatase) by IFCC method done. Statistical Analysis Used: Tabulation and Pie Circle Result: 193 of the total 229 vaccinated HCWs tested positive for core antibody, meaning that they were infected prior to HBs Ag vaccination, leaving a total of 36 'truly' vaccinated HCWs. 11 HBs Ag positive HCWs were tested for Liver Profile and all had ALAT, ASAT and ALP within normal range. Out of total number of 141 HCWs having 10 and below IU/L anti HBs, 5 HCWs were positive for HBS Ag, showing a positivity of 3.5%. Conclusion: Need of vaccination and for post-vaccination serological testing of all HCWs considering the high rates of non-responders and low responders (anti-HBs-34.2%). Importance of educating the HCWs of safety precautions while handling body fluids, and the management of ' sharps ' injuries.

Mycobacterium kumamotonense is a novel, slow-growing non-chromogenic nontuberculous mycobacterium, which belongs to Mycobacterium terrae complex. We report, for the first time in Greece, the isolation of M. kumamotonense from an immunocompetent patient with pulmonary infection and latent tuberculosis. M. kumamotonense was identified by sequencing analysis of 16S rDNA and 65-kDa heat shock protein genes while by commercial molecular assays it was misidentified as Mycobacterium celatum. Antibiotic susceptibility testing was performed by the reference broth microdilution method. The strain was susceptible to amikacin, clarithromycin, rifampin, ciprofloxacin, moxifloxacin, rifabutin, ethambutol and linezolid.

Scrub typhus is emerging as an important cause of acute febrile illness in Northern India. This is a report of two sisters presenting concurrently with acute respiratory distress syndrome. A diagnosis of scrub typhus was made in both the patients, and they were successfully treated with doxycycline.