The specimens were collected from the neonatal intensive care unit of the IPGMER and SSKM Hospital, Kolkata, India from 2007(January) to 2014(June). Due to some unavoidable circumstances specimens could not be collected from January 2012 to June 2012. This unit has a 16 bed Level III unit, 26 bed Level II unit and 8 bed neonatal surgical units. This unit had 1185 admissions per year (departmental census 2013), including both intramural and extramural births. Blood cultures of the neonates having sepsis were processed by previously described methods (Roy et al., 2013 (link)). In brief, 1 ml of blood for culture was drawn with aseptic precautions from a peripheral vein into Peds Plus vials. Blood culture was performed with a BACTEC9050 system (Becton Dickinson, Sparks, MD, USA) from 2008 onwards and manually prior to that. For any culture that flagged positive, Gram-staining was performed and subculture was done on appropriate media based on Gram's stain: MacConkey agar (Difco Laboratories, Detroit, MI, USA) and 5% sheep blood agar (Difco Laboratories) for Gram-negative and Gram-positive organisms, respectively. Clinical data were collected from the hospital registers.
Bactec9050 system
Manufactured by BD
Sourced in United States
The BACTEC9050 system is a fully automated blood culture instrument designed for the detection and monitoring of microbial growth in blood samples. The system utilizes specialized culture bottles and growth detection technology to facilitate the identification of microorganisms in a clinical laboratory setting.
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5 protocols using bactec9050 system
1
Neonatal Sepsis Surveillance Protocol
2
Bacterial Culture Procedures for Pediatric Sepsis
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Bacterial cultures were performed as previously described [39 (link)]. Briefly, blood cultures were performed for all children at enrollment in which ~1.0 mL of venipuncture blood was collected aseptically and directly inoculated into the pediatric blood culture bottle (Peds Plus, Becton-Dickinson) that were incubated in an automated BACTEC 9050 system (Becton-Dickinson), for 5 days. Positive cultures were examined by Gram staining and sub-cultured on blood agar, chocolate agar or MacConkey agar plates based on the Gram stain results. Bacterial isolates were identified according to standard microbiologic procedures as described previously [39 (link)]. According to the Kenya Ministry of Health national guideline [45 ], empirical antimicrobial therapy was initiated for all children with suspected bacterial infection using oral and/or injections which included combination of cloxacillin/ampicillin, chloramphenicol, ciprofloxacin/norfloxacin/nalidixic acid, ceftriaxone, gentamicin, penicillin, metronidazole or doxycycline. Children with severe malnutrition were treated with penicillin/gentamicin plus metronidazole. Antibiotic therapy was reviewed according to the results of the blood cultures.
3
Bloodstream Infections by K. pneumoniae in Indian Neonates
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During 2014 to 2016, among a total of 285 culture-positive cases, 107 (2014, n = 19; 2015, n = 42; 2016, n = 46) non-duplicate K. pneumoniae were recovered from blood of septicemic neonates admitted to the level III unit (Neonatal intensive care unit) of an Indian tertiary care hospital. Blood specimens from the sick neonates were collected at the hospital as part of the routine diagnostic procedure. As a standard protocol, 1 mL of blood for culture was drawn with strict aseptic precautions from a peripheral vein of neonates suspected with sepsis, and inoculated in BD Bactec Peds Plus Vial (Becton Dickinson, MD, USA). Blood culture was performed with the automated Bactec 9050 system (Becton, Dickinson). Gram staining was performed for any culture that flagged positive, and the subculture was carried out on appropriate media based on Gram’s stain: MacConkey agar and 5% sheep blood agar (Difco Laboratories, Detroit, MI, USA) for Gram-negative and Gram-positive isolates, respectively. Bacterial identification and antibiotic susceptibility were performed by Vitek-2 compact system and disk diffusion (as per CLSI guidelines) (35 ), respectively.
4
Quantifying Bacterial Burden in Blood Cultures
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BD BACTEC Plus Aerobic Blood Culture Bottles (Fisher Scientific) were inoculated with 9 mL of human whole blood (with sodium citrate as the anti-coagulant) purchased from Lampire Biological Laboratories Inc. (Catalog No. 7203706). The blood was tested at Lampire using their standard quality control process. A 0.5 McF of the test organism was prepared from colonies and diluted before spiking the blood culture bottle to produce a final concentration of 2–3 CFU/mL. Bottles were incubated in the BD BACTEC 9050 System until flagged as positive by the instrument. PBCs were then diluted in saline (Hardy Diagnostics) and plated on blood agar for colony counts before the eQUANT method was performed. The drop count method was used, in which seven 10 uL drops were evenly placed onto a blood agar plate which was incubated in air at 35 °C for 18–24 h. Following incubation, the number of colonies in each drop was counted and the dilution factor was applied to calculate the CFU/ml concentration of the PBC.
5
Comprehensive Clinical Microbiology Protocols
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Aerobic and anaerobic blood culture vials (Becton–Dickinson, Franklin Lakes, NJ, USA) were incubated in the automated BD Bactec 9050 system for a maximum of 5 days or until the culture became positive. Standard culture-based methods were used for species identification [API Test stripes (bioMérieux, Craponne, France) and BBL Enterotubes or BBL Oxi/Ferm Tube (Becton–Dickinson, Franklin Lakes, NJ, USA)]. Coagulase-negative staphylococci and Bacillus spp. were routinely considered contaminants. Streptococcus viridians were regarded contaminants as well, unless the patient had clinical signs of endocarditis or meningitis. As part of a clinical trial requirement, the microbiology laboratory at the ASH successfully participates in regular external quality assurance programs addressing species identification.
For HIV testing, a rapid test was used [Vikia HIV 1/2 (bioMérieux, Craponne, France), or DetermineTM HIV 1/2, (Alere, Yavne, Israel), depending on local availability]. In case of a positive reading, the result was confirmed by VIDAS HIV DUO Ultra (bioMérieux, Craponne, France) and Immunocomb HIV1&2 Bispot (Alere, Yavne, Israel). CD4 counts were done using BD FACS count (Becton–Dickinson, Franklin Lakes, NJ, USA). The Lambaréné method to analyze thick smears was applied to diagnose malaria [11 (link)].
For HIV testing, a rapid test was used [Vikia HIV 1/2 (bioMérieux, Craponne, France), or DetermineTM HIV 1/2, (Alere, Yavne, Israel), depending on local availability]. In case of a positive reading, the result was confirmed by VIDAS HIV DUO Ultra (bioMérieux, Craponne, France) and Immunocomb HIV1&2 Bispot (Alere, Yavne, Israel). CD4 counts were done using BD FACS count (Becton–Dickinson, Franklin Lakes, NJ, USA). The Lambaréné method to analyze thick smears was applied to diagnose malaria [11 (link)].
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