DOI 10.

1007/s11094-017-1644-x
Pharmaceutical Chemistry Journal, Vol. 51, No. 6, September, 2017 (Russian Original Vol. 51, No. 6, June, 2017)

LIMIT OF DETECTION OF MICROORGANISMS

IN DRUG STERILITY TESTING

M. V. Roshchina,1 O. V. Gunar,1 and N. G. Sakhno1

Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 51, No. 6, pp. 62 – 64, June, 2017.

Original article submitted April 7, 2017.

The use of compendial and alternative methods for drug sterility testing, the most important characteristic of
which is the limit of detection (LOD), i.e., the minimum amount of viable microorganisms that can be found in
the examined sample, is currently regulated abroad. The goal of the research was to determine experimentally
and compare the LODs of microorganisms by direct inoculation and using a BacT/ALERT 3D Microbial De-
tection System automated bacteriological analyzer. Statistically significant differences between the results ob-
tained by the different methods were not found.
Keywords: drugs, sterility testing, limit of detection, method sensitivity.

One of the main tasks of the pharmaceutical industry is
assessing the quality and safety of medicines on the con-
sumer market. Sterility [1] and microbiological purity [2] are
some of the most important quality parameters of medicines.
Currently, sterile drugs represent a significant fraction of the
Russian Federation pharmaceutical market and play an im-
portant role in the therapy of various diseases.
The US and European pharmacopoeias [3, 4] allow the
use of alternative microbiological methods aimed at obtain-
ing faster, more accurate, more sensitive, and reproducible
results in addition to direct inoculation and membrane filtra-
tion methods for drug sterility testing. In 2006, the Center for
Drug Evaluation and Research (CDER) of the USA pub-
lished an article with evidence that new microbiological
methods had indisputable advantages with respect to speed
and accuracy of determining drug quality [5].
Herein, the methods most suitable for quality analysis of
unfiltered medicines, i.e., direct inoculation and an alterna-
tive based on colorimetric determination of CO2 in the cultu-
re medium, were compared.
Microorganisms (aerobic and anaerobic bacteria, yeast-
like and mold fungi) that are actively multiplying in an ap-
propriate liquid culture medium utilize certain nutrients and
form metabolic products such as CO2 and other metabolites.
1
Scientific Centre for Expert Evaluation of Medicinal Products, Ministry of
Public Health of the Russian Federation, 8/2 Petrovskii Blvd., Moscow,
127051, Russia.
The amount of CO2 produced in closed vials could be de-
tected and used as an indicator of growing microorganisms.
The CO2 level determined by a sensor depends on the
initial microorganism concentration, i.e., the higher the ini-
tial concentration, the faster the detection.
This principle was implemented in instruments such as
BacT/ALERT (Biomerieux, France), BACTEC (Becton
Dickinson, USA), and VersaTREK (Thermo Fisher Scien-
tific, USA).
Sterility testing is a quality method. Therefore, if several
examined samples include just one nonsterile unit (ampule,
vial, etc.), this is necessary and sufficient to establish the
noncompliance of the batch of medicines to the Sterility pa-
rameter. The goal of sterility testing is to confirm the total
exclusion of viable microorganisms in the analyzed drug. The
probability of finding contamination increases, as a rule, with
increasing number of microbial cells in the examined sample
and varies depending on the type of microorganism [6].
The limit of detection (LOD) is one of the most impor-
tant characteristics that must be compared for the rapid and
reference methods to assess objectively the potential of an al-
ternative microbiological method. The LOD in microbiology
is the minimum number of microorganisms that can be deter-
mined [7].
The goal of the present work was to establish and com-
pare the LODs of microorganisms using direct inoculation on
growth media [8] and an automated BacT/ALERT 3D Micro-
bial Detection System (Biomerieux, France).

508
0091-150X/17/5106-0508 © 2017 Springer Science+Business Media New York
Limit of Detection of Microorganisms 509

LODs are difficult to establish experimentally because 2. Standard BioBall samples (Biomerieux, France) with
all microbiological methods are highly variable. Successive di- 104 CFU for B. subtilis NCTC 10400, S. pyogenes NCTC
lutions followed by inoculations of growth media with several 12696, P. acnes DSM 1897, P. aeruginosa NCTC 12924, and
concentrations of microorganisms provided reliable data [9]. C. albicans NCPF 3179 and 550 CFU for C. sporogenes
NCTC 12935 dissolved in a special liquid for rehydration.
EXPERIMENTAL BIOLOGICAL PART 3. Prepared spore suspension of B. subtilis ATCC 19659
(Merck, Germany) of concentration 107 CFU/mL.
LODs were determined using test strains of aerobic and Successive dilutions produced suspensions of test strains
anaerobic bacteria and yeast-like fungi (Table 1). with theoretical cell contents of 50, 10, 5, 1, 0.5, and 0.05
The following growth media were used to determine the
CFU/mL.
LODs:
The accuracy of the dilutions (positive control) was con-
for direct inoculation, liquid thioglycolic medium was
firmed and the actual cell contents in the suspensions were
used to identify aerobic and anaerobic bacteria; tryptic soy
established by inoculating a dilution (0.1 mL) of 102
broth (TSB) and liquid Sabouraud medium, yeast-like and
mold fungi. CFU/mL on the surface of agar growth media. Grown colo-
for the automated BacT/ALERT 3D system, SA (me- nies were counted after 24 – 48 h.
dium based on TSB) was used to identify aerobic microor- Sample inoculation. An inoculate (1 mL) with the re-
ganisms (bacteria and fungi), SN (medium based on TSB), quired concentration of each microorganism in 10-fold repli-
anaerobic microorganisms. cations was placed under aseptic conditions into vials and
Preparation of inoculate. The work used: tubes. The tubes were incubated for 14 d in thermostats at
1. 24-hour cultures of microorganisms grown on slanted 32.5 ± 2.5°C for bacteria and 22.5 ± 2.5°C for C. albicans.
dense growth media (P. aeruginosa ATCC 9027, S. aureus Inoculated vials were placed into the incubation module of
ATCC 6538, E. faecalis BKM B-602, E. hirae BKM B-2107,
the BacT/ALERT 3D instrument and cultivated for 7 d at
A faecalis SCPM 300205, K. rhizophila ATCC 9341, and
35.0 ± 0.5°C.
C. sporogenes ATCC 19404, on tryptic soy agar; L.
Results were observed and counted daily. Tubes were ex-
plantarum BKM B-578, on lactobacillus agar; C. albicans
amined in transmitted light to determine microorganism
ATCC 10231, an Sabouraud agar with chloramphenicol) and
were rinsed off with sterile saline (0.9% NaCl). Then, micro- growth. Growth was measured by the turbidity, film forma-
bial suspensions of bacteria with a cell concentration of 109 tion, or precipitation. The BacT/ALERT 3D system performed
CFU/mL were prepared using a WHO turbidity standard (10 automated monitoring with detection of contaminated vials
U); of C. albicans, 107 CFU/mL. and construction of microorganism growth curves.
The generalized Spearman—Karber formula [10] was
used for statistical data processing. The 50% LOD (LOD50)
was calculated. This parameter characterized the number of
TABLE 1. Microorganism Test Strains
microorganism cells that could be identified in 50% of the in-
Microorganism Strain No.
stances. Confidence intervals (CIs) were also calculated for
Aerobic bacteria the calculated LOD50 values.
Bacillus subtilis ATCC 19659, NCTC 10400
Pseudomonas aeruginosa ATCC 9027, NCTC 12924
Staphylococcus aureus ATCC 6538
TABLE 2. Limit of Detection (LOD50) of Microorganisms and
Streptococcus pyogenes NCTC 12696 Confidence Intervals (CIs)
Alcaligenes faecalis GKPM 300205 Direct inoculation, BacT/ALERT 3D,
Test strain
Kocuria rhizophila ATCC 9341 CFU/10 mL CFU/10 mL
Anaerobic bacteria B. subtilis 35.5 (21.6 – 58.2) 3.5 (2.3 – 5.3)
Clostridium sporogenes ATCC 19404, NCTC 12935 S. aureus 12.6 (10.5 – 15.1) 5.2 (3.3 – 8.1)
Propionibacterium acnes DSM 1897 P. aeruginosa 1.32 (1.10 – 1.58) 2.8 (2.0 – 3.8)
Enterococcus faecalis BKM B-602 S. pyogenes 91.6 (65.4 – 128.2) 5.1 (3.6 – 7.4)
Enterococcus hirae BKM B-2107 K. rhizophila 28.2 (17.9 – 44.2) 27.2 (15.3 – 48.4)
Lactobacillus plantarum BKM B-578 L. plantarum 44.6 (26.0 – 76.7) 15.3 (10.2 – 23.0)
Yeast-like fungi A. faecalis 14.1 (8.2 – 24.3) 13.6 (8.6 – 21.4)
Candida albicans ATCC 10231, NCPF 3179 C. albicans 12.8 (10.7 – 15.4) 4.0 (2.8 – 5.8)
510 M. V. Roshchina et al.

TABLE 3. Limit of Detection and Confidence Intervals for Obli- number of E. hirae whereas there was no substantial differ-
gate and Facultative Anaerobic Bacteria ence for E. faecalis. Anaerobic bacteria P. acnes and C. spo-
rogenes did not grow on SA growth medium whereas the
Direct BacT/ALERT 3D
inoculation sensitivity of SN special medium was an order of magnitude
(liquid greater than the values obtained by direct inoculation.
Test strain SA medium, SN medium,
thiogylcolic The main accuracy characteristic, i.e., the Fisher crite-
medium), CFU/10 mL CFU/10 mL
rion (F), the calculated values of which were less than the ta-
CFU/10 mL
ble values (Fcalc.max = 18.09 < Ftable = 19.00), was deter-
C. sporogenes 7.1 HP <1 mined in the work. This indicated that statistically significant
P. acnes 73.2 HP 7.8 differences between the results from the different methods
(52.3 – 102.5) (5.0 – 11.9) were not observed.
E. hirae 17.8 (11.3 – 27.9) 15.5 8.3
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