Rare Cases of Ralstonia pickettii Associated with Tonsillitis and Wound Infection

Background:      We describe here two rare case of Ralstonia pickettii infection associated with tonsillitis and wound infection. Ralstonia pickettii is a gram negative bacteria, usually isolated from the soil and water. Infection-associated Ralstonia species though rare, has become an emerging nosocomial pathogen due to its capability to survive in harsh conditions like antiseptic solutions. Both of our reported cases presented to the outdoor patient unit and thus possibility of community acquired Ralstonia picketti infection cannot be ruled out. Methods:   Isolates were subjected to Siemens MicroScan WalkAway 96 Plus (Beckman Caulter Diagnostics, USA) for identification and antimicrobial susceptibility testing. Results:  Ralstonia pickettii was identified as a rarely detected pathogen. Conclusion:    Ralstonia pickettii though rarely associated with wound infections and tonsillitis, could be a potential hospital acquired pathogen

Comparative evaluation of IS6110 PCR via conventional methods in rapid diagnosis of new and previously treated cases of extrapulmonary tuberculosis

ÖZET Yeni ve önceden tedavi edilmiş ekstrapulmoner tüberkülozlu hastaların hızlı tanısında IS6110 ile konvansiyonel yöntemlerin karşılaştırılmalı değerlendirilmesi Gelişmekte olan ülkelerde ekstrapulmoner tüberküloz (EPTB) tanısı önemli bir problemdir. EPTB'de, az sayıda basil içerme özelliği, yetersiz miktarda örnek gibi birçok sorun bulunmaktadır. Bütün bu kısıtlamalar, konvansiyonel bakteriyolojik tekniklerin EPTB tanısına düşük katkısına neden olmaktadır. Nükleik asit amplifikasyon yöntemleri, mikobakteriyel DNA'nın saptanması amacıyla geliştirilen hızlı ve duyarlı tekniklerdir. Mycobacterium tuberculosis complex'in spesifik genomunda yer alan "insertion sequence" IS6110'a ait 123bp'nin DNA fragmanı, EPTB'nin hızlı tanısı amacıyla polimeraz zincir reaksiyonu (PCR) ile çoğaltıldı. Bu çalışmada, yeni ve önceden tedavi edilmiş EPTB'li hastaların hızlı tanısında IS6110 PCR ile konvansiyonel yöntemler karşılaştırıldı. EPTB şüpheli hastalardan 450 örnek toplandı ve Mycobacteria için Zeihl Neelson (ZN) boyama ve M. tuberculosis için BACTEC kültürü yapıldı. Bütün örnekler ayrıca, M. tuberculosis complex'in insertion element IS6110'un 123bp fragmanını hedefleyen primerlerle PCR amplifikasyonu ile IS6110 için çalışıldı. Testler arasında duyarlılık bakımından anlamlı fark saptandı. Dört yüz elli örnek . Bununla birlikte, testler arasında spesifisite bakımından anlamlı fark yoktu (p> 0.05). IS6110 PCR'nin hem yeni hem de önceden tedavi edilmiş hastalarda, yayma mikroskopi ve BACTEC kültüründen daha duyarlı olduğunu bulduk. IS6110 PCR, yeni ve önceden tedavi edilmiş EPTB'li hastaların tanısında kullanışlı olabilir. Şüpheli EPTB'li hastaların tedavi kararında fayda sağlayabilir. Anahtar Kelimeler: Tüberküloz, ekstrapulmoner tüberküloz, polimeraz zincir reaksiyonu, IS6110. Yazışma Adresi (Address for Correspondence): Dr. Surya KANT, Chhatrapati Shahu Ji Maharaj Medical University UP (Erstwhile King George Medical College), LUCKNOW -INDIA e-mail: [email protected] Tuberculosis (TB) continues to be a major global public health problem. Incidence of extrapulmonary tuberculosis (EPTB) is on increasing worldwide as well as in India (1,2). EPTB compromises 20% of all TB cases in India (3). Diagnosis of EPTB in different clinical presentations has been always as challenge. Smear microscopy and culture lack of sensitivity in EPTB case and culture (solid and liquid media) also takes at least two to four weeks for grow of mycobacteria. A study has reported smear positive is around 10-37% of the patients and mycobacterial culture is positive in variable proportional 12-80% in different biological specimens (3). Studies from many laboratories around the global were using primers most commonly targeting the IS6110 insertion element (4-9). The detection of the IS6110 insertion element present in form of multiple copies to detect of Mycobacterium tuberculosis complex but not other mycobacterial species (9-11). Polymerase chain reaction (PCR) using IS6110 insertion sequences as the target, has potential to conquer limitation of conventional method and to established as rapid, sensitive technique for detecting DNA of M. tuberculosis in different clinical specimens from respiratory and non respiratory sites MATERIALS and METHODS Study Design The study was performed prospectively in a blinded manner. Clinical Specimens and Data Collection 2-5 mL of specimens was collected from 450 specimens, non-repeated specimens from suspected cases of extrapulmonary tuberculosis. The specimens were included as Lymph Node Aspirate and Cold Abscesses, Pleural fluid, C.S.F, Synovial Fluid, Ascetic Fluid, Urine, Gastric Aspirate, Pus, Bone Marrow, Wound and Pus swab and Others specimens (biopsies tissues). All specimens were kept in ice box and transported Mycobacteriology Laboratory, Department of Microbiology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, India for smear examination by ZN Staining, BACTEC Culture and PCR test. All patients were signed with due informed consent of the patients from indoor and outward wards of Department of Pulmonary Medicine, Chhatrapati Shahuji Maharaj Medical University, Lucknow, India and Mycobacteriology Laboratory, Department of Microbiology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, India during Jan 2009 to Dec 2010. The clinical history regarding, present and past history of antitubercular treatment (ATT); family history of tuberculosis and any other associated disease were taken in prescribed Performa. Microbiological Analysis of Extra Pulmonary Specimens Specimens was divided in to two part one part was kept at -20 for PCR till processing and another part was processed for mycobacterial smear preparation and BACTEC culture. Smears were stained with Ziehl Neelsen (ZN) method and examined for acid-fast bacilli (AFB) (21). BACTEC vials were incubated and interpreted as per Becton Dickinson (BD, Sparks, MD, USA) manual instructions (22). NAP (p-nitro-α-acetylamino-β-hydroxy propiophenone) (Becton Dickinson, Sparks, MD, USA), identification was done to differentiate M. tuberculosis form non tuberculous mycobacteria (22). A decrease or unchanged growth index (GI) in nap vial indicated presence of M. tuberculosis complex (MTBC), while an increase in GI indicated the presence of Mycobacterium other than tuberculosis (MOTT). Standard H 37 Rv strain of M. tuberculosis complex was used as positive control. Extraction of DNA Extraction of DNA was done by the CTAB (cetyl-trimethyl-ammonium bromide) -phenol chloroform extraction method (23). Specimens were centrifuged at 10.000 rpm for 10 min. The supernatant was discarded and the pellet suspended in 567 µL of TE (Tris EDTA, pH 7.4) buffer, 30 µL 10% SDS (sodium dodecyl sulfate) and 3 µL proteinase K (20 mg/mL), mixed and incubated at 37°C for 1 hour. After incubation, 100 µL of 5 M NaCl and 80 µL of high-salt CTAB buffer (containing 4 M NaCl, 1.8% CTAB was added and mixed followed by incubation at 65°C for 10 min. An approximate equal volume (0.7-0.8 µL) of chloroform-isoamyl alcohol (24.1) was added, mixed thoroughly and centrifuged for 4-5 min in a microcentrifuge at 12.000 rpm. The aqueous viscous supernatant was carefully decanted and transferred to a new tube. An equal volume of phenol: chloroform-isoamyl alcohol (1:1) was added followed by a 5 min spin at 12.000 rpm. The supernatant was separated and then mixed with 0.6 volume of isopropanol to get a precipitate. The precipitated nucleic acids were washed with 75% ethanol, dried and re-suspended in 100 µL of TE buffer. Primer and IS6110 PCR The amplification reaction was performed in a final volume of 20 µL. the reaction mixture contained 10 µL Pyrostart Fast PCR Master Mix 2X (dNTP, Taq polymerase with Mgcl 2 , Fermentas, India), 1 µL (10 pmole) of each primer, 3 µL water (nuclease free) and 5 µL of extracted DNA. The oligonucleotide primers used were IS1 and IS2, are: 5'-CCT GCG AGC GTA GGC GTC GG3' and 5' CTC GTC CAG CGC CGC TTC GG 3' respectively (SBS Gentech Co. Ltd) (24). These primers amplified a target fragment at 123 base pairs (bp) from the insertion, M. tuberculosis sequence element IS6110. The PCR amplification was done in thermal cycler (MJ Research, PTC-100, GMI, Inc, USA), which involved 40 cycles of denaturation at 94°C for 2 minute, annealing of primers at 68°C for 2 minute, and primer extension at 72°C for 1 minute. The amplified products were separated on 2% agarose gels, visualized on a UV-light transilluminator (Bangalore Genei, Bangalore, India). The presence of 123bp fragment indicate as positive test as M. tuberculosis complex. The positive controls included the DNA of H37Rv strain. Negative control included PCR grade water Statistical Analysis Data were analyzed using SPSS 15.0 (Statistical Package for the Social Sciences, Chicago, IL, USA) for Maurya AK, Kant S, Nag VL, Kushwaha RAS, Kumar M, Dhole TN. 215 Tüberküloz ve Toraks Dergisi 2011; 59(3): 213-220 Windows. The significance of difference was taken as significance value (p< 0.05).Sensitivity was calculated as [Tp/(Tp + Fn)] x 100; specificity was calculated as [Tn/(Tn + Fp)] x 100; Tp = total number of positives; Tn = total number of negatives; Fp = total number of false positive, Fn = total number of false negative; respectively. RESULTS Specimen's Characterization of Extrapulmonary Tuberculosis Cases During the two year study period, 470 clinical specimens were strong clinical suspicion of extrapulmonary tuberculosis were subjected from tertiary care hospitals and all mention test were performed. Out of these, 20 specimens found to be contaminated in BACTEC culture. 450 specimens of results were used in the study. Out of 450 specimens, 153 (34%) lymph node aspirate and cold abscesses, 58 (12.8%) pleural fluid, 44 (9.7%) cerebrum spinal fluid (CSF), 48 (10.7%) urine, 31(6.8%) ascetic fluid, 26 (5.8%) pus, 22 (4.9%) wound and pus swab, 16 (3.5%) gastric aspirate, 10 (2.2%) bone marrow, 10 (2.2%) synovial fluid and 30 (6.7%) others specimens (biopsies tissues). Out of 450 patients, 320 (71.1%) patients were males and 130 (28.9%) females. The mean age of all patients was 39.8 ± 16.1 years. Patients 25-44 years of age accounted for 45% of the total cases. Out of 450 cases, 328 (72.8%) were new cases and 122 (22.2%) were previously treated cases of EPTB. Detection Rate of M. tuberculosis by IS6110 PCR, BACTEC Culture and ZN Smear Microscopy According to New Cases and Previously Treated Cases All specimens were colleted from suspected case of extra pulmonary tuberculosis were found to be AFB positive were 60 (13.4%). On the basis of cases, we found that sensitivity of AFB staining on EPTB were 37 (11.2%) in new cases and 23 (18.8%) in previously treated cases. The sensitivity of AFB staining was higher in comparison to previously treated cases. Overall detection rate of M. tuberculosis by AFB Staining was 60 (13.4%). The detection of M. tuberculosis by BACTEC culture was 202 (45%). Results of BACTEC culture according to cases, 151 (46.03%) were in new cases and 51 (41.8%) were in previously treated cases. We found that sensitivity of BACTEC culture was higher in new cases. All culture isolates obtained were confirmed as mycobacteria with biochemical tests mentioned. Using IS 6110 PCR, 283 (61.8%) were positive for IS6110 PCR for M. tuberculosis. 203 (61.8%) were positive in new cases and 80 (65.5%) were positive in previously treated cases. We found that sensitivity of IS6110 PCR was higher in previously treated cases. Overall comparison of tests, IS6110 PCR was found to have much higComparative evaluation of IS6110 PCR via conventional methods in rapid diagnosis of new and previously treated cases of extrapulmonary tuberculosis 216 Tüberküloz ve Toraks Dergisi 2011; 59(3): 213-220 Comparison of Sensitivity of IS6110 PCR Test Via Others Conventional Tests According to New Cases and Previously Treated Cases IS6110 PCR test was found to be much more sensitive than ZN staining and BACTEC culture results individually as well as in combination are shown in 217 Tüberküloz ve Toraks Dergisi 2011; 59(3): 213-220 DISCUSSION Tuberculosis (TB) is a major public health dilemma in India. India is the highest TB burden country accounting for one fifth of the global incidence. Global annual incidence estimate is 9.4 million cases out of which it is estimated that 1.98 million cases are from India (26). In India, EPTB comprises 20% of all TB cases. Its prevalence in the country varies between 8.3-13.1% in different districts according to cohort analysis by Central TB Division, Ministry of Health and Family Welfare in 2002 (27,28). The diagnosis of extrapulmonary tuberculosis is till now challenging for diagnostic routine laborites. Numeric reasons are showing that, lack of adequate specimens amounts or volumes; distribute of the specimens for different diagnostic tests (histology/cytology, biochemical analysis, microbiology, and PCR), non-uniform distribution of microorganisms; paucibacillary nature of the specimens; presence of inhibitors that undermine the performance of nucleic acid amplification-based techniques; and the lack of an efficient sample processing technique universally applicable on all types of extrapulmonary samples (29). The poor performance of conventional M. tuberculosis detection techniques, based on microscopic examination of Ziehl-Neelsen stained and culture of M. tuberculosis (LJ Medium and BACTEC Radiometric culture) are still in widespread use for diagnostic purposes, still though they fail to provide the required sensitivity and specificity. The PCR test would be particularly useful in the diagnosis of EPTB where conventional microbiological techniques for M. tuberculosis are showing poor performance of sensitivity. The specificity, sensitivity and speed of PCR test in diagnosis of M. tuberculosis infection shown in this study should encourage the use of this method in routine diagnosis of EPTB. Previously studies shown the success of microscopy is highly variable from 22% to 96% and most authors rate it at round 60% (30-32). Our results shown that sensitivity of smear microscopy was 13.7% and specificity was 100%. The sensitivity of microscopy depends on the clinical presentation and more than 10.000 bacilli per milliliter are necessary for secure microscopic positivity (33). Our studies shown that conventional bacteriological technique were positive in 202 (45%) specimens, where as IS6110 PCR showed that 283 (63%) specimens were positive for M. tuberculosis. The difference was found that to be statistical significant (p< 0.05). Several studies have been reported on PCR to detect M. tuberculosis (34-39). The detection of the IS6110 insertion element present in multiple copies to detect M. tuberculosis complex, but not other mycobacterial species 218 Tüberküloz ve Toraks Dergisi 2011; 59(3): 213-220 tion and PCR results were positive but BACTEC culture was negative; these could be the presence of nonviable mycobacteria in the sample as patients were receiving antitubercular treatment. IS6110 PCR test is higher sensitivity than microscopy and the culture and could help in therapeutic decision for patients with clinical suspicion of EPTB. CONCLUSION IS6110 PCR test for DNA specific M. tuberculosis may be hopes of a rapid and accurate diagnostic test for EPTB and it will help where conventional diagnosis fails and provisional diagnosis of tuberculosis is made on the basis of clinical presentation and histology/cytology examination without evidence of AFB. IS6110 PCR may be great potential to improve the clinician vision for the early diagnosis, treatment and prevention of EPTB. ACKNOWLEDGEMEN

Clinical outcome, viral response and safety profile of chloroquine in COVID-19 patients — initial experience

Introduction: Chloroquine and its analogues are currently being investigated for the treatment and post exposure prophylaxis of COVID-19 due to its antiviral activity and immunomodulatory activity.Material and methods: Confirmed symptomatic cases of COVID-19 were included in the study. Patients were supposed to receive chloroquine (CQ) 500 mg twice daily for 7 days. Due to a change in institutional protocol, initial patients received chloroquine and subsequent patients who did not receive chloroquine served as negative controls. Clinical effectiveness was determined in terms of timing of symptom resolution and conversion rate of reverse transcriptase polymerase chain reaction (RT-PCR) on day 14 and day 15 of admission.Results: Twelve COVID-19 patients formed the treatment arm and 17 patients were included in the control arm. The duration of symptoms among the CQ treated group (6.3 ± 2.7 days) was significantly (p-value = 0.009) lower than that of the control group (8.9 ± 2.2 days). There was no significant difference in the rate of RT-PCR negativity in both groups. 2 patients out of 12 developed diarrhea in the CQ therapy arm.  Conclusion: The duration of symptoms among the treated group (with chloroquine) was significantly lower than that of the control group. RT-PCR conversion was not significantly different between the 2 groups

Response surface methodology-based optimisation of agarose gel electrophoresis for screening and electropherotyping of rotavirus

Management of rotavirus diarrhoea cases and prevention of nosocomial infection require rapid diagnostic method at the patient care level. Diagnostic tests currently available are not routinely used due to economic or sensitivity/specificity constraints. Agarose-based sieving media and running conditions were modulated by using central composite design and response surface methodology for screening and electropherotyping of rotaviruses. The electrophoretic resolution of rotavirus genome was calculated from input parameters characterising the gel matrix structure and running conditions. Resolution of rotavirus genome was calculated by densitometric analysis of the gel. The parameters at critical values were able to resolve 11 segmented rotavirus genome. Better resolution and electropherotypic variation in 11 segmented double-stranded RNA genome of rotavirus was detected at 1.96% (w/v) agarose concentration, 0.073 mol l<SUP>-1</SUP> ionic strength of Tris base-boric acid-ethylenediamine tetraacetic acid buffer (1.4×) and 4.31 h of electrophoresis at 4.6 V cm<SUP>-1</SUP> electric field strength. Modified agarose gel electrophoresis can replace other methods as a simplified alternative for routine detection of rotavirus where it is not in practice

Dermatophytoses and spectrum of dermatophytes in patients attending a teaching hospital in Western Rajasthan, India

Introduction: About 20–25% of the world's populations are infected with dermatophyte and the incidence of the disease is increasing steadily. In India, the cause of dermatophytoses is adversely influenced by economic factors such as poverty, poor hygiene and social conditions like overcrowding. The main objective of this study was to determine the prevalence, etiological agents, and distribution patterns of the disease among the patients attending a teaching hospital, Jodhpur. Materials and Methods: This is a retrospective study conducted in the Department of Microbiology and Department of Dermatology of a teaching hospital, Jodhpur from January 2017 to May 2018. The microbiological records of potassium hydroxide (KOH) mount examination and the fungal culture report of skin, hair, and nail samples during the study period were analyzed. Results: A total 363 skin, hair, and nail samples were received for fungal culture as investigations requested by dermatologist. Out of total 363 samples, KOH mount was positive in 58.4% and the culture was positive in 44.07% cases. Dermatophyte was isolated in 40.22% cases. Trichophyton mentagrophytes was the predominant dermatophyte (55%) isolated followed by T. tonsurans (22.5%) and T. rubrum (6.25%). Conclusion: The present study gives an insight about the prevalence and distribution pattern of dermatophytoses in Jodhpur, India. Tinea corporis was the most commonly diagnosed clinical condition followed by Tinea cruris. T. mentagrophytes was implicated as the predominating species

Stenotrophomonas maltophilia mimicking Klebsiella on Chromogenic Media

Chromogenic media are being used now-a-days for identification of many microorganisms in the clinical microbiology laboratories. They are being used for screening purposes especially in case of urine cultures, environmental surveillance and also for detection of multidrug-resistant organisms like Methicillin-resistant Staphylococcus aureus (MRSA), Vancomycin-resistant Enterococci (VRE) and Carbapenem-resistant Enterobactericeae (CRE) etc. Organisms produce specific colour due to digestion of chromogenic substrates present in the medium. Thus the Organisms are identified on basis of colour production. These media are specific in detecting common pathogens which are isolated. But they can give different results when uncommon organisms are encountered. In this case report the uncommon organism that is Stenotrophomonas maltophilia produced colour similar to Klebsiella spp. on chromogenic media

An observational study on carbapenem-resistant Enterobacterales (CRE) colonisation and subsequent risk of infection in an adult intensive care unit (ICU) at a tertiary care hospital in India

Summary: Background: Carbapenem-resistant Enterobacterales (CRE) are a global health problem with a growing prevalence. India has a high prevalence of CRE. CRE infections are difficult to treat, and are associated with significant morbidity and mortality. Colonisation is generally a prerequisite for infection and the prevention of CRE colonisation is key to the prevention of CRE infection. Objectives: To determine the prevalence of CRE colonisation and subsequent infections in an adult intensive care unit (ICU) in India. Methods: We conducted a prospective observational study in which perirectal swabs were obtained along with relevant clinical details of consenting adult patients upon ICU admission between January 2019 and August 2020. Rectal screening was performed using MacConkey agar plates with ertapenem disks and further identification was performed using conventional microbiological techniques. Ertapenem minimum inhibitory concentration (MIC) was determined using an epsillometer (E) test. The modified carbapenem inactivation (mCIM) test and EDTA carbapenem inactivation test (eCIM) were performed to confirm carbapenem resistance using the Clinical Laboratory Standards Institute (CLSI) 2020 guidelines. Results: 192 ICU patients were screened for CRE. 37 patients were found to be colonised with CRE. Klebsiella pneumoniae (N=25; 67.6%) was the most frequent CRE isolate, followed by Escherichia coli (N=11; 29.7%) and one Enterobacter species (N=1; 2.7%). 89.2% (33/37) patients developed CRE infection. Pneumonia was the most common CRE infection identified in 12/33 (36.4%) patients.during the hospital stay. The median duration of hospital stay was longer (17 days) for CRE colonised compared to CRE non-colonised patients (9 days) (P<0.001). Death occurred in 27 % (N=10/37) of CRE-colonised patients during the hospital admission. Conclusion: CRE colonisation is associated with high risk of subsequent CRE infection and longer ICU and hospital admission

Nursing students: A vulnerable health-care worker for needlesticks injuries in teaching hospitals

Background: Occupational exposure to bloodborne pathogen is a significant risk to health-care workers. In any teaching hospital apart from regular health-care workers and employees, there are significant population of students and trainee. It is important to assess the health-care worker in hospital which has maximum chances of exposure to these pathogens. The aim of this study is to determine the most susceptible job group for needlestick injury (NSI) reported in a newly established teaching medical institute in the Western part of Rajasthan, India. Methods: This is a retrospective analysis of data of NSI occurred during September 2014 to January 2017. Results: Sixty three NSIs were reported during the study. Nursing students were the most vulnerable group who reported maximum number of NSI. Among the nursing students, 72% were completely vaccinated against hepatitis B virus. Conclusions: Nursing students are at utmost risk for NSIs, the prevention of which requires regular training and education

Frequency of OXA-Type Carbapenemases among Carbapenem-Resistant <i>Acinetobacter baumannii</i> in Clinical Isolates from Adult Intensive Care Unit in India

Purpose Acinetobacter baumannii is a highly virulent bacteria in modern health care, with a high ability to acquire antimicrobial resistance. Carbapenemases production appears to be the most common mechanism involved in drug resistance to carbapenem. As the prevalence of carbapenem-resistant A. baumannii was high in intensive care unit (ICU) patients, this study was designed to find the frequency of oxacillinases (OXA) genes including OXA-23, OXA-24, OXA-51, and OXA-58