In vitro assessment of newer colistin-sparing antimicrobial agents for clinical isolates of carbapenem-resistant organisms.

OBJECTIVES: Carbapenem-resistant organisms (CROs) are a significant public health threat globally, particularly in countries like India with high antibiotic resistance rates. The current study investigates the prevalence of CROs, detects resistance mechanisms using phenotypic methods and assesses the efficacy of newer antibiotics against CROs. METHODS: A prospective study conducted at a tertiary care hospital in India during 2021-23. Clinical specimens were processed and bacterial identification was performed using MALDI-TOF mass spectrometry followed by antimicrobial susceptibility testing using CLSI guidelines against a plethora of newer antibiotics for CROs. Carbapenemase production was detected using phenotypic methods, and the presence of the mcr-1 gene was assessed by real-time PCR. RESULTS: During the study period, predominantly (70 %) Gram-negative bacteria were isolated; amongst which 5692 strains were carbapenem-resistant, wherein resistance to different carbapenems ranged from 34.1% to 79 %. Majority of the carbapenemase producers were metallo-ß-lactamases (MBL) producers (75 %). Colistin resistance was noted in 5.4 % of selected carbapenem-resistant isolates. Among newer antibiotics, cefiderocol demonstrated the lowest resistance rates (0-14 %), while resistance to newer ß-lactam/ß-lactamase inhibitor combinations was very high in carbapenem-resistant isolates. Fosfomycin, minocycline and tigecycline, each showing variable efficacy depending on the site of infection. Moreover, newer ß-lactam/ß-lactamase inhibitor combinations offer restricted benefits due to widespread prevalence of MBL in the region. CONCLUSION: The escalating prevalence of CROs in India underscores the urgency for alternative treatment options beyond colistin. Hence, highlighting the critical importance of developing effective strategies to combat carbapenem resistance.

Possible impact of revisions in disc diffusion breakpoints for aminoglycosides and piperacillin/tazobactam in the 33rd edition of CLSI M100 document on clinical reporting and use in Indian settings with low susceptibility.

PURPOSE: The study explores the impact of significant interpretative breakpoint changes for aminoglycosides and piperacillin-tazobactam in Enterobacterales and Pseudomonas aeruginosa, considering PK/PD, clinical data, and susceptibility on clinical reporting and use. PROCEDURE: Between January 2021 and June 2023, a total of 189,583 samples were processed for bacterial pathogens and antimicrobial susceptibility testing was performed using disc diffusion method/VITEK® 2 Compact system/broth microdilution. WHONET software was utilised to capture and analyse the changes in the interpretation of disc diffusion method, following updates to CLSI M100 documents in comparison to previous editions. Antimicrobial consumption data was collected and interpreted as DDD/100 bed days using AMC tool software. Here, we present data for 13,615 members of Order Enterobacterales and 1793 Pseudomonas aeruginosa isolates. FINDING: Enterobacterales exhibited a significant susceptibility drop of 14.7% for gentamicin and 21.7% for amikacin. Pseudomonas aeruginosa showed an increase in isolates with intermediate tobramycin susceptibility, from 0.6% to 29.7%, with relatively minor changes in piperacillin-tazobactam interpretation. CONCLUSION: The changes indicate a shift toward increased 'resistance' and 'intermediate susceptibility' for these antibiotics, emphasizing the need for cautious use and leveraging PK/PD knowledge for improved antibiotic utilization, patient outcomes, and antimicrobial stewardship.

Molecular Characterization of New Delhi Metallo-ß-Lactamases-Producing Bacteria in the Gastrointestinal Tract of Intensive Care Unit Patients.

BACKGROUND: The emergence of carbapenem-resistance in the gut flora of patients in the intensive care unit (ICU) poses a significant risk for infection with these types of pathogens. MATERIALS AND METHODS: New Delhi metallo-ß-lactamase 1 (NDM-1) in the gut flora of ICU patients was detected in cultures of a single rectal swab from each patient admitted to the ICU for a minimum period of 48 hrs. Samples were processed in the microbiology laboratory using blood agar and MacConkey agar. Identification of pathogens, carbapenem resistance, and metallo-ß-lactamase production was made using standard laboratory procedures. Bacterial isolates were also used for the determination of the NDM-1 gene by molecular methods. RESULTS: One hundred twenty-two patients with different clinical presentations were recruited in the study. Two hundred nine bacteria were isolated, with Escherichia coli being the most common isolate. A total of 54/122 (44.3%) patients harbored carbapenem-resistant organisms (CRO), 36/122 (29.5%) carried metallo-ß-lactamase-producing organisms (MBLO), and 30/122 (24.6%) carried bacteria with the NDM-1 gene. Patients who harbored CRO and MBLO had longer mean duration of stay in the ICU and hospital than those not harboring CRO and MBLO. All the metallo-ß-lactamases were simultaneously resistant to other groups of antibiotics also. Use of invasive devices, three or more classes of antibiotics, hospitalization during the previous six months, comorbidities, and hospital stay for ≥48 hours before ICU admission had a significant association with colonization with CRO. CONCLUSION: Patients admitted in ICU or with serious diseases should be screened for gastrointestinal carriage of carbapenem-resistant organisms. Irrational use of antibiotics must be stopped to prevent the emergence and spread of such organisms.

Correlation between Venereal Diseases Research Laboratory titers and CD4 T-lymphocyte count determined by flow cytometry in HIV-infected adults: A 5-year study.

INTRODUCTION: Venereal Diseases Research Laboratory (VDRL) is one of the key tests for the diagnosis of syphilis; however in HIV-positive individuals, it has been reported to give inappropriate results at times. Thus, this study was conducted to determine if the VDRL test titers vary with the severity of immunosuppression as determined by CD4 cell count. MATERIALS AND METHODS: A total of 2630 samples from HIV-positive adults were tested by qualitative and quantitative VDRL test and if reactive, by Treponema pallidum hemagglutination (TPHA) test. CD4 cell counts were determined at the same time by flow cytometry (BD FACSCount™ system). Correlation between CD4 T-lymphocyte cell count and VDRL titers was sought for. RESULTS: Nearly 6.7% (176/2631) of individuals were VDRL reactive, males more than females (7.6% vs. 5.1%, P = 0.014). Four of the VDRL-reactive patients were found negative by TPHA test and were excluded from further study. VDRL titers ranged from weakly reactive to being reactive at 1:128 (median = 1:2). The CD4 cell count ranged from 23 cells/µl to 883 cells/µl (median = 276 cells/µl, mean = 323.9 ± 200.9). Pearson's coefficient of correlation (R) between CD4 cell count and VDRL titers was calculated to be 0.0559; coefficient of determination (R2) was 0.0031. CONCLUSIONS: Although the correlation coefficient shows a positive correlation, the association was very weak. Therefore, CD4 cell count cannot be expected to influence VDRL titers in HIV-positive adults significantly.

Statistical analysis of microbiological diagnostic tests.

No study in medical science is complete without application of the statistical principles. Incorrect application of statistical tests causes incorrect interpretation of the study results obtained through hard work. Yet statistics remains one of the most neglected and loathed areas, probably due to the lack of understanding of the basic principles. In microbiology, rapid progress is being made in the field of diagnostic test, and a huge number of studies being conducted are related to the evaluation of these tests. Therefore, a good knowledge of statistical principles will aid a microbiologist to plan, conduct and interpret the result. The initial part of this review discusses the study designs, types of variables, principles of sampling, calculation of sample size, types of errors and power of the study. Subsequently, description of the performance characteristics of a diagnostic test, receiver operator characteristic curve and tests of significance are explained. Lack of a perfect gold standard test against which our test is being compared can hamper the study results; thus, it becomes essential to apply the remedial measures described here. Rapid computerisation has made statistical calculations much simpler, obviating the need for the routine researcher to rote learn the derivations and apply the complex formulae. Thus, greater focus has been laid on developing an understanding of principles. Finally, it should be kept in mind that a diagnostic test may show exemplary statistical results, yet it may not be useful in the routine laboratory or in the field; thus, its operational characteristics are as important as the statistical results.

Utility of Laboratory Diagnosis for Confirmation of the Syndromic Case Management in Married Indian Women with Vaginal Discharge.

OBJECTIVE: WHO and NACO recommend treatment of STIs/RTIs on the basis syndromic case management (SCM), even without laboratory confirmation, which may lead to over-treatment. Thus, this study was conducted to evaluate the utility of laboratory diagnosis for confirmation of patients with vaginal discharge diagnosed on the basis of SCM. METHODOLOGY: 234 married women in reproductive age group, diagnosed as having vaginal discharge syndrome were included. Normal saline wet-mount slide preparations were made for detection of motile trichomonads. Gram stained smear were prepared and scored as per classification developed by Nugent. The presence of pseudohyphae and/or budding yeast cells was considered diagnostic of candidal infection. VDRL, TPHA and HIV testing were also done as per protocol. RESULTS: The median age of the study population was 34 years. Most common cause was bacterial vaginosis (positive= 21.4%, 95% CI= 16.6-27.1%; intermediate score= 17.5%, 95% CI= 13.2-22.9%), followed by candidiasis (13.7%, 95% CI= 98-18.7%) and trichomoniasis (0.4%, 95% CI= 0-2.6%). No etiological diagnosis for vaginal discharge could be established in approximately half of the women. Only two women were HIV positive; one was reactive by VDRL and TPHA tests. CONCLUSION: Our study highlights the possible lacunae in SCM. Large number of patients may be over-treated if only syndromic management is followed, with financial, medical and social implications. Thus we recommend, the treatment maybe initiated on the basis of SCM, but it is essential that laboratory diagnosis is sought for and the treatment modified accordingly.

Co-Infections and Sero-Prevalence of HIV, Syphilis, Hepatitis B and C Infections in Sexually Transmitted Infections Clinic Attendees of Tertiary Care Hospital in North India.

BACKGROUND: HIV, syphilis, hepatitis B and C (HBV & HCV) infections modify the epidemiology and presentation of each other. This study aimed to estimate the seroprevalence of these infections and their co-infections in sexually transmitted infections (STI) clinic attendees in New Delhi, India. METHODS: A retrospective study including 220 patients was conducted during May 2014 through December 2014. Serodiagnosis of HIV was performed as per Strategy III of NACO guidelines; syphilis by VDRL followed by TPHA; HBV and HCV by rapid immuno-chromatographic test followed by ELISA. RESULTS: Male subjects were slightly more in number as compared to females (56.36% vs. 43.63%). Twelve (5.45%), 14 (6.36%), three (1.36 %) and one (0.45%) were reactive for HIV, VDRL, HBV and HCV, respectively. Three were both HIV and syphilis positive and one was both HIV and HBV positive; no co-infections of HBV/HCV, HIV/HBV/HCV and HIV/HBV/HCV/syphilis coexisted. CONCLUSIONS: High prevalence of HIV, HBV, HCV and syphilis in STI clinic attendees mandate routine screening to detect co-infections and follow prompt therapy in order to minimize their sequelae.

Sexually transmitted infections and HIV in self reporting men who have sex with men: A two-year study from India.

Homosexuality is not legally and socially accepted in India. Thus, this area of research has largely been ignored by Indian authors, resulting in dearth of knowledge, particularly with respect to sexually transmitted infections (STIs) in this high-risk group. Over a period of two years (2013-2014), 738 males sought care at skin and venereal diseases clinics, 52 (7.05%, 95% CI=5.4-9.14%) of who identified themselves as MSM and were enrolled in the study. Diagnosis was made on the basis of clinical presentation and laboratory testing, wherever indicated. Thirty six percent of MSM had only homosexual preferences, while 64% were bisexual. The most common sexually transmitted infection was genital warts (23.08%, 95% CI=13.58-36.28%). Fourteen patients (26.92%, 95% CI=16.67-40.35%) were VDRL and TPHA positive (two, five and four with primary syphilis, secondary syphilis and latent syphilis, respectively). These were followed by genital herpes (11.54%, 95% CI=5.03-23.34%), genital molluscum contagiosum (9.62% 95% CI=3.75-21.04%), and gonorrhea (5.77%, 95% CI=1.38-16.25%). Of those tested, 23.08% (95% CI=13.58-36.28%) of patients were reactive for HIV serology. Thus, MSM is a high-risk group with high prevalence of HIV and other STIs in this group, mandating greater focus, education and counseling.

Multi drug resistance and Extended Spectrum Beta Lactamases in clinical isolates of Shigella: A study from New Delhi, India.

BACKGROUND: Shigella is an important cause of gastroenteritis in local Indian population, as well as of traveler's diarrhea in the international visitors to India. These patients often require appropriate antimicrobial therapy; however, rapid development of antimicrobial resistance poses a major hurdle in achieving this goal. METHOD: A prospective study was conducted during 2009-12 in New Delhi, India, including 6339 stool samples from gastroenteritis patients. 121 Shigella strains were identified on the basis of colony morphology, biochemical reactions, serotyping and ipaH gene based PCR. Antimicrobial susceptibility testing by disc diffusion, MIC determination by Vitek(®) 2 and phenotypic tests for ESBL/AmpC production were done. RESULTS: Nineteen percent strains (23/121) were found to be resistant to third generation cephalosporins and all were phenotypically confirmed to be ESBL producers; one strain was positive for AmpC. ESBL producing strains were also found to be significantly more resistant (p < 0.05) to several other antimicrobials agents in comparison to ESBL non-producers, [ampicillin (100% vs. 62.2%), ampicillin/sulbactam (100% vs. 30.6%), cotrimoxazole (100% vs. 77.6%), ciprofloxacin (87.0% vs. 49.0%), ofloxacin (87.0% vs. 52.0%) and gentamicin (30.4% vs. 7.1%)]. Multidrug resistance was seen in 76% strains. CONCLUSIONS: Inappropriate use of antimicrobial agents puts high selection pressure on the higher-end antibiotics. Multi-drug resistance and high rates of ESBL production by Shigella is a matter of concern for the local population as well as international travelers. Therefore, better national level antimicrobial management programs are the priority needs.

True Prevalence of Shigellosis in Indian Children with Acute Gastroenteritis: Have We Been Missing the Diagnosis?

BACKGROUND: Shigella is responsible for high morbidity and mortality among children, yet its true prevalence remains inconclusive. The aim of this study was to determine the actual prevalence of Shigella infection in childhood diarrhea and dysentery cases and assess the applicability of ipaH gene PCR in Indian settings. METHODS: This study was conducted at Maulana Azad Medical College and associated Lok Nayak Hospital, New Delhi, India during 2011-12. A total of 385 children (207 with diarrhea, 118 with dysentery, and 60 matched controls) were enrolled. Stool samples were cultured, and the suspected colonies were analyzed using biochemical reactions and serotyping. Antimicrobial susceptibility testing was done using disc diffusion method. ipaH-gene PCR was performed directly on stool samples collected from 180 randomly selected patients (60 from each group). RESULTS: Shigella was isolated using conventional culture methods in 8.2% (95% CI: 5.1%, 12.8%), 33.1% (95% CI: 25.2%, 42.0%), and 0% in the diarrhea, dysentery and control cases, respectively. High resistance was seen towards co-trimoxazole, nalidixic acid, fluoroquinolones, doxycycline and several beta-lactams drugs. Actual prevalence of shigellosis was determined using ipaH gene PCR to be 18.3% (95% CI: 10.4% - 30.1%) diarrhea cases and 56.7% (95% CI: 44.1, 68.4%) dysentery cases. One (1.7%, 95% CI: 0.01%, 9.7%) control specimen also yielded positive result in PCR. CONCLUSIONS: Correct diagnosis of shigellosis is essential to start antimicrobial therapy in selected cases. The prevalence of Shigella / EIEC infection in children is much higher than previously estimated. Despite its high costs and other limitations, we recommend the use of ipaH-gene PCR as a routine tool in the management of childhood acute gastroenteritis cases.

A comparative study of bacterial and parasitic intestinal infections in India.

BACKGROUND: Infectious diarrhea causes a major health problem in developing countries with significant morbidity and mortality. Very often, rehydration therapy alone does not suffice, mandating the use of antimicrobial agents. However, rapidly decreasing antimicrobial susceptibility is complicating the matters. MATERIALS AND METHODS: The study aimed to determine the prevalent bacterial and parasitic agents of diarrhea in India. A cross-sectional study was done at Maulana Azad Medical College and associated Lok Nayak Hospital, New Delhi, during 2012-14. Stool samples were received from patients of all age groups and processed for bacteriological and parasitological identification by microscopy, bacterial culture, biochemical identification, serotyping and antimicrobial susceptibility tests. The study also aimed to identify the recent papers (after year 2000) reporting aetiology of infectious diarrhea in India involving the general population as a whole and compare them with present findings. RESULTS: Out of 6527 samples, 581 (8.90%) were positive for bacterial pathogens. A total of 280 samples (of 3823 under-five year children) were positive for diarrheagenic Escherichia coli. Other organisms like Vibrio cholera were found in 159 (2.44%) cases, Shigella spp. in 126 (1.93%), Salmonella Typhi in 7 (0.11%), Salmonella Typhimurium in 6 (0.10%), Aeromonas hydrophila in 3 (0.05%) cases. Levels of resistance to nalidixic acid, amoxicillin and ciprofloxacin were alarmingly high. Third generation cephalosporins were seen to be moderately active except against E. coli. Parasites were identified in 312 (4.78%) cases. Giardia intestinalis, Ascaris lumbricoides and Entamoeba histolytica were identified in 2.27%, 1.15% and 0.64% cases respectively. CONCLUSION: Analysis of recent nationwide studies revealed V. cholerae was the most common bacterial/parasitic agent of diarrhea across all populations, being followed by diarrheagenic E. coli and Giardia intestinalis. Periodic laboratory monitoring of antimicrobial susceptibility pattern is essential, as is formulation of effective antibiotic use policy.

A comparative study of conventional and molecular techniques in diagnosis of campylobacter gastroenteritis in children.

BACKGROUND: Campylobacter species are a significant cause of gastroenteritis among children worldwide. Conventional methods for detection of Campylobacter spp. based on cultural isolation and biochemical tests are cumbersome and time consuming. Because of their superior sensitivity and cost effectiveness, molecular methods are often used for identification of the pathogens. AIMS: To evaluate different diagnostic methods for identification of Campylobacter. MATERIALS AND METHODS: Faecal samples were collected from 585 children (age ≤ 12 years) with acute diarrhoea admitted in a tertiary-care hospital, excluding children already on antimicrobial therapy. All samples were examined by four methods: Grams' staining, culture methods, Enzyme-Immuno Assay, and Polymerase Chain Reaction (PCR). After Grams' staining, samples were inoculated on modified charcoal cefoperazone deoxycholate agar. ProSpecT™ Microplate Assay® and PCR assay using cadF gene was done for detection of Campylobacter specific antigen and DNA, respectively, in faecal samples. McNemar's test was used to compare the results wherever applicable. RESULTS: 197 cases (33.67%) were found to be positive for Campylobacter by at least one method. But only 121 (20.78%) out of the 585 stool specimens tested fulfilled the positivity criteria, i.e., positive either by culture or by any two tests among other three. Culture had very low sensitivity (37.19%), whereas PCR had the highest (96.69%) sensitivity but lowest positive predictive value (86.03%). Rapid Grams' staining technique (sensitivity 63.64%) was found to be better than culture. Detection by PCR and ELISA was significantly better than by culture on selective media and Grams' staining (p<0.0001). CONCLUSIONS: Molecular techniques significantly increased detection rates of Campylobacter in children with diarrhoea. However, enzyme-immuno assay with high accuracy has the advantage of applicability in resource-poor settings.

Enteric pathogens, immune status and therapeutic response in diarrhea in HIV/AIDS adult subjects from north India.

Intestinal infection causing diarrheal disease is a dominant contributor to high morbidity and mortality in developing countries. This intervention study aimed to assess the response of specific anti-microbial and anti-retroviral therapy (ART) on enteropathogens identified in HIV/AIDS adult subjects from northern India. Seventy five ART naive (group 1) and seventy five ART adherent (group 2) HIV/AIDS adult subjects with diarrhea were enrolled. Stool samples from all subjects were examined for enteropathogens by wet mount, staining methods, culture and ELISA. Subjects with enteropathogens were started on specific therapy as per National AIDS Control Organisation, Government of India's guidelines. Follow-up stool samples were examined after 2-4 weeks of completion of therapy for persistence/clearing of enteropathogens. CD4+ T lymphocyte count was done for all subjects. At enrollment, group 1 had 26.13% bacterial, 57.66% parasitic & 16.22% fungal pathogens while group 2 had 11.9%, 69.05% & 19.05% pathogens, respectively. Parasitic diarrhea was more common than bacterial diarrhea. The coccidian parasites (Cryptosporidium spp. & Isospora belli) were the common parasites identified. Clearance of enteric pathogens was significant after specific anti-microbial therapy (p = 0.0001). Persistence of enteropathogens was seen primarily for coccidian parasites. Clearance of enteropathogens after specific therapy and the diagnostic yield of stool specimens were influenced by the CD4+ counts. Immune competence coupled with specific anti-microbial therapy displays the best response against enteric pathogens.

Increasing antimicrobial resistance of campylobacter jejuni isolated from paediatric diarrhea cases in a tertiary care hospital of new delhi, India.

INTRODUCTION: The resistance to the clinically important antimicrobial agents, particularly the fluoroquinolones and the macrolides, is increasing among the Campylobacter isolates. Only limited data is available regarding the changing antimicrobial resistance pattern in the Indian scenario. METHODOLOGY: Three hundred fifty cases (ages ≤12years) of acute diarrhoea, who were admitted to a tertiary-care hospital, were investigated for Campylobacter spp. The antimicrobial susceptibilities of all the C. jejuni isolates were assessed by the disk diffusion method according to the CLSI guidelines. RESULTS: A total of 36 isolates of C. jejuni were tested for their antimicrobial susceptibilities. A high degree of resistance to the fluoroquinolones (100% to Nalidixic acid and 86.66% to Ciprofloxacin) was detected in the Campylobacter isolates. The frequency of resistance against Tetracycline was 33.33% and that against Erythromycin was 22.2%. Fifteen (41.66%) isolates were multiresistant, being resistant to 3 or more antimicrobial agents. CONCLUSIONS: An increased resistance to the quinolones and the macrolides and multidrug resistance warrant a reconsideration of their use as the drugs of choice in patients with severe gastroenteritis when Campylobacter is the presumed cause.

Clinical and Microbiological Profile of HIV/AIDS Cases with Diarrhea in North India.

Intestinal infections are a significant cause of morbidity and mortality in people living with HIV/AIDS (PLWHA) especially in developing countries. The present study was conducted to assess the clinical and microbiological spectrum in HIV/AIDS cases with diarrhea and to correlate the occurrence of such pathogens with stool characters, HIV seropositivity status, and CD4 counts. Stools from 154 HIV seropositive subjects and 50 HIV negative controls were examined by direct microscopy, fecal cultures, and serological tests (Clostridium difficile Toxin A, Cryptosporidium antigen, and Entamoeba histolytica antigen ELISA). CD4 T cell enumeration was done using FACS count (Becton Dickinson). The study showed a male preponderance (112 males and 42 females). Weakness, abdominal pain, and anorexia were the most common symptoms. Coccidian parasites were the most common cause of diarrhea in HIV seropositive cases. C. parvum was seen in 60.42% while Isospora belli in 9.03%. Amongst the bacterial pathogens C. difficile was detected in 18.06%, diarrheagenic Escherichia coli in 11.11%, and Shigella spp. in 2.78%. Pathogen isolation rates were more in HIV seropositive cases and subjects with low CD4 T lymphocyte counts. Regular monitoring of CD4 T lymphocyte counts and screening for enteric pathogens will help improve the quality of life for PLWHA.