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10]

Original Article

Cross‑Country Transport and Isolation and Identification of

Streptococcus pneumoniae By Use of Alternate Sources of
Blood Supplemented Media among Laboratories in India
Satish Kumar Amarnath, Sangeeta Joshi1, Madhuwanti N. Abhyankar2, Ranjeeta Adhikary1, H. B. Beena1, T. D. Chugh3, K. D. Gandhi4, Vivek Hittinahalli5,
V. A. Indumathi6, Mukhopadhyay Rajavari7, S. Muralidharan8, S. S. Rao9, I. Roy10, N. Saini11
Medical Director, Manipal Clinics, 1Consultant Microbiologist, Manipal Hospital, 5Consultant Microbiologist, Yashomati Hospital, 6M.S. Ramaiah Medical College,
8
St. John’s Medical College Hospital, Bengaluru, Karnataka, 2Consultant Microbiologist, Golwilkar Metropolis Health Services, (I) Pvt. Ltd., Pune, 9SS Microbiology
Laboratory, Thane, Maharashtra, 3Sr. Consultant, Department of Microbiology, BL Kapoor Memorial Hospital, 4Consultant Microbiologist, Shanti Mukund Hospital,
11
Consultant Microbiologist, Pushpanjali Hospital, New Delhi, 7Consultant Microbiologist, West Bank Hospital, Howrah, 10Consultant Microbiologist, Sri Aurobindo Seva
Kendra, Kolkata, West Bengal, India

Abstract
Background: The isolation of S. pneumoniae (Sp) depends on specimen integrity / transport, media and expertise. The non-availability of
sheep blood agar poses a challenge in identification of colonial morphology and identification in India. Methods: Laboratories processed
swabs containing either pure Sp or Sp in mixed cultures with a second (confounding) bacterium shipped across the country in cold conditions.
Duplicate set of swabs was shipped back to the central laboratory to assess the impact of shipping on culture viability. The identical swab
was cultured on sheep, human blood and one additional agar plate used in the laboratory. Results: 46/60(77%) of cultures containing only Sp
were correctly identified. In specimens where Sp was present in mixed culture, the proportion of isolates in which Sp was correctly identified
varied, with most variability attributed to the particular confounding organism rather than the media. There was no discernible impact of
temperature-controlled (4-6°C) transport on the isolation of Sp from culture swabs. Conclusions: The study clearly elucidates the ability of
laboratories for isolation of S. pneumoniae on human blood agar in resource limited settings. The results highlight the difficulties inherent
in correctly identifying pathogens in mixed cultures in needs improvement using standardized tests across the study centers. The study also
reaffirms the ability to transport biological specimens over long geographical distances without loss.

Keywords: Blood supplementation, efficacy, isolation, Streptococcus pneumoniae, transport under cold conditions

Introduction alternative sources of blood are used in the preparation of blood

Microbiology laboratory science is predicated upon the use of
standardised methods to accurately identify and characterise
pathogens. The gold standard for identification of bacterial
pathogens remains isolation of viable organisms by culture
and often requires a source of blood as a culture medium
supplement. [1] Growth of bacteria on blood agar plates
allows for characterisation of the pathogen based on colony
morphology, haemolytic patterns and identification tests.[2‑43]
Supplementation of agar media with sheep’s blood is
the standard method for cultivation of Streptococcus
pneumoniae  (SP). In many developing countries, however,
sheep’s blood is not readily available, and locally available
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DOI:
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agar media. A commonly used supplement is human blood,
which is sourced from blood banks. The use of human blood
agar has been criticised because many pathogenic bacteria
exhibit altered growth and haemolytic patterns when grown
Address for correspondence: Dr. Satish Kumar Amarnath,
Manipal Clinics, The Annexe, 98/1 Rustom Bagh, HAL (Old) Airport Road,
Bengaluru ‑ 560 017, Karnataka, India.
E‑mail: satish.amarnath@manipalhospitals.com
Received : 04-08-2019 Revised : 04-09-2019
Accepted : 04-11-2019 Published Online : 05-12-2019
This is an open access journal, and articles are distributed under the terms of the Creative
Commons Attribution‑NonCommercial‑ShareAlike 4.0 License, which allows others to
remix, tweak, and build upon the work non‑commercially, as long as appropriate credit
is given and the new creations are licensed under the identical terms.
For reprints contact: reprints@medknow.com
How to cite this article: Amarnath SK, Joshi S, Abhyankar MN,
Adhikary  R, Beena HB, Chugh TD, et al. Cross-country transport and
isolation and identification of Streptococcus pneumoniae by use of alternate
sources of blood supplemented media among laboratories in India. Indian
J Med Microbiol 2019;37:363-9.

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Amarnath, et al.: Isolation and identification of S. pneumoniae after cross‑country transport
on agar plates prepared from human blood rather than animal
blood, which may result in errors in pathogen identification.[1,4]
This altered growth and haemolysis may be due to differences
in red cell morphology and membrane composition, which
impact lysis by bacterial haemolysins.[2,8,9,16,21,22,27,29‑37,39] The
human blood may expose the laboratory worker to blood‑borne
pathogens, have inhibitory antibodies and antibiotics which
may impact isolation of SP. To date, there have been few
studies assessing the performance of laboratories in identifying
pneumococcus based on the source of blood used for blood
agar.[2,3]
There is an acknowledged need for improving the quality and
capacity of developing world laboratories to identify common
bacterial pathogens. Characterisation of variability among
laboratories in their ability to successfully identify common
pathogens is one step towards advancing standardised
laboratory practices that will lead to more reliable diagnosis
and improved patient care.[7]
Here, we report a field study, in which we assessed the
ability of microbiology laboratories in India to reliably detect
pneumococcus using several locally available types of blood
agar media and compared the use of sheep blood agar. In
addition, we evaluated the effect of transport on the viability
of cultures and laboratory performance.
Materials and Methods
Bacterial strains and media
The bacterial strains used were as follows: SP (ATCC49619),
Streptococcus pyogenes  (ATCC19615), Streptococcus
agalactiae (ATCC12386), Streptococcus mutans (ATCC35668),
Staphylococcus epidermidis  (ATCC12228) and Moraxella
catarrhalis (ATCC817). All isolates were obtained from the
American Type  Culture Collection  (Manassas, VA, USA).
The selection of bacteria was made based on common
pathogens isolated in respiratory specimens, mimicking
colony morphology and haemolysis patterns. These were
grown in brain–heart infusion broth (BHI) and resuspended
in BHI supplemented with skim milk and glycerol prior to
aliquoting and storing at  −70°C. Participating laboratories
were supplied with both sheep blood agar (SBA) and human
blood agar  (HBA) plates obtained from Fitech Chemitron
Laboratories (Bengaluru, India). In addition, each participating
Table 1: Blood agar sources used in the study
Medium Heavy Moderate Scanty Total
Human BA* 6 4 5 15
Biomeriuex BA 1 1 1 3
Delta BA 2 2 1 5
Hi‑media BA 1 1 1 3
Sheep EOS BA 0 1 0 1
Sheep BA (*) 5 4 9 18
Total 15 13 17 45
*The significance – Produced locally in the reporting laboratory. BA:
Blood agar
364 Indian Journal of Medical Microbiology  ¦  Volume 37  ¦  Issue 3  ¦  July-September 2019
laboratory utilised an agar plate of their selection as a
comparator [Table 1].
Rabbit plasma, Optochin and Bacitracin disks  (all from
Hi‑media Laboratories, Mumbai, India) and bile esculin
medium (Fitech Chemitron, Bengaluru, India) were supplied
to the laboratories to ensure uniformity in the performance of
biochemical tests.
Study design
Ten laboratories  (study sites) in five metropolitan
cities (Bengaluru, Delhi, Kolkata, Mumbai and Pune) across
India were participated. The laboratories selected included
seven hospital‑based laboratories (one medical college), two
diagnostic laboratories and one quality control laboratory.
All were large facilities responsible for the routine culture of
bacterial pathogens. A central laboratory (Manipal Hospital
Microbiology Laboratory, Bengaluru, India) prepared the
culture swabs  (described below) and other study materials
for delivery to each study site. Each study site was shipped
two identical sets of 34 culture swabs per batch; one was
cultured at the local site, while the other was returned to the
central laboratory  (to examine the effects of transportation
on the samples). Study sites were required to culture each
of the 34 provided swabs on HBA and sheep blood agar
(provided in a blinded fashion by the central laboratory) and
one agar routinely used by the study site [procured/produced
individually by each study site, Table  1]. This resulted in
102 inoculated agar plates for each study site.
This study was conducted in accordance with the principles
of Good Clinical and Laboratory Practices and was approved
by the appropriate institutional review boards and ethics
committees at each site as well as by the ethics committee at
Manipal Hospital. CTRI registration was not done since there
were no human subjects in the study.
Preparation of culture swabs
Swabs were prepared so that each swab contained either SP
alone or a combination of SP with a confounding bacterial
species. Frozen  (−70°C) bacterial stocks were thawed and
subcultured overnight on sheep blood agar and then the
bacteria were resuspended in BHI broth. During the pilot
study, 0.5 MacFarlands concentration of each microorganism
was made and diluted and standardised based on the growth
on SBA done by the quadrant method. Heavy indicated
growth on all four quadrants, moderate indicated growth in
the first and second quadrants and scanty indicated growth
was when the growth was in the primary inoculum site only.
Various combinations of the organisms were made using
different concentrations of SP and confounding bacteria
(moderate growth) [Tables 2 and 3]. Swabs provided along
with Amies Transport Media (Hi‑media Laboratories, Mumbai,
India) were dipped in the suspension and then gently squeezed
on the sides of the tube to expel the extra broth and stabbed
into the transport medium. Each batch was made up of 34 swab
sets in duplicate and readied for dispatch.
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Amarnath, et al.: Isolation and identification of S. pneumoniae after cross‑country transport

Table 2: Number of cultures performed at individual study sites

Confounding bacterium
SP concentration None S. pyogenes S. mutans M. catarrhalis S. epidermidis S. agalactiae
High 6 3 3 3 3 3
Medium 6 3 3 3 3 3
Scanty 6 3 3 3 3 3
None 9 6 6 6 6 6
S. pneumoniae: Streptococcus pneumoniae, SP: Streptococcus pneumoniae, S. pyogenes: Streptococcus pyogenes, S. mutans: Streptococcus mutans,
M. catarrhalis: Moraxella catarrhalis, S. epidermidis: Staphylococcus epidermidis, S. agalactiae: Streptococcus agalactiae

Table 3: Percent recovery of isolates from either pure or mixed culture swabs
Bacteria Recovery of SP (n=60) Recovery of confounding bacterium (n=50)
SBA HBA Local agar SBA HBA Local agar
SP alone 77 77 77 ‑ ‑ ‑
SP + S. pyogenes 33 37 67 27 20 3
S. pyogenes alone ‑ ‑ ‑ 30 25 25
SP + S. agalactiae 13 3 20 37 27 33
S. agalactiae alone ‑ ‑ ‑ 35 25 40
SP + M. catarrhalis 67 73 60 23 17 3
M. catarrhalis alone ‑ ‑ ‑ 40 30 30
SP + S. epidermidis 47 43 63 30 33 67
S. epidermidis alone ‑ ‑ ‑ 45 50 100
SP + S. mutans 77 60 80 0 13 0
S. mutans alone ‑ ‑ ‑ 10 10 10
SBA: Sheep blood agar, HBA: Human blood agar, S. pneumoniae: Streptococcus pneumoniae, SP: Streptococcus pneumoniae, S. pyogenes: Streptococcus pyogenes,
S. agalactiae: Streptococcus agalactiae, M. catarrhalis: Moraxella catarrhalis, S. epidermidis: Staphylococcus epidermidis, S. mutans: Streptococcus mutans

The 102 swabs were shifted in 3 batches of 34 swabs per batch
with 21 SP with 15 of them in combination with confounding
organism  (3 per species), 10 pure cultures of confounding
organism only (2 per species) and 3 containing no organisms.
This gives a final figure of 63 SP with 45 of them being in
combination with confounding organism, 30 having pure
growth of confounding organism and 9 with no organism in
the study.
These swabs were prepared in duplicate sets. The swabs were
immediately kept in a 2°C–6°C cold room before dispatch.
Pilot testing
The simulated swabs were made in batches of 7 and kept
in a cold room at temperature controlled conditions to test
the viability of SP under storage conditions. It was seen that
the organisms survived in Amies transport media for 4 days
without loss of viability and concentration. This helped us to
plan the dispatch with temperature controlled boxes from Blue
Dart Temperature Controlled Logistics which assured us that
shipments would be maintained between 2°C and 6°C for more
than 48 h.[3‑7,10‑15,17‑20,23‑28,38,40‑43]
Media/swab distribution and testing
A total of three sets with 34 swabs each were sent across
with HBA and SBA media. Sets of 34 specimen swabs were
bundled together. This bundle along with its duplicate was
put in separately in the consignment. Each consignment was
packed by 4.00 PM and 5.00 PM on Monday, the consignment
left the central laboratory to the destinations. The Bengaluru
deliveries were also subjected to the same process, thereby
duplicating the sample transportation schedules of the
outstation deliveries, and in turn, ensuring that all laboratories
received the material.[13]
The receiving laboratories subcultured the appropriate swab
on to the coded blood agar and reported the specimen as a
routine respiratory specimen. All the duplicate swabs returned
to the central laboratory were subcultured onto SBA. The
laboratories were asked to record the organisms into a data
sheet and record the isolated organisms. A digital photograph
of the primary growth to record the growth of the organisms
for cross verification was done. Laboratories were asked
to report the cultures within 72 h of receipt using standard
techniques.[14,18]
At the outset, we received the duplicate set of swabs from each
centre on Wednesday, which was double the time it took to
transship the specimens across the country. The reason we sent
the full complement of swabs is to ensure that each swab with its
bacteria could withstand the temperature of transshipment to every
location. The swabs were subcultured on SBA on receipt in the
central laboratory and reported. The central laboratory took special
care to see that the SP was obtained in the concentrations sent
across, and it duly photographed the medium for documentation.
The transportation temperature was monitored by digital data
loggers both to and from the study centres.[19] Once the reports

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Amarnath, et al.: Isolation and identification of S. pneumoniae after cross‑country transport
were received the principal investigator decoded and analysed
the results.
Statistics
Data from the centres were pooled and analysed using Epi‑info
version  6.02 software  (Centers for Disease Control and
Prevention), Atlanta, USA.
Results
Specimen isolation and identification
The performance of all the blood agars was evaluated
separately. The source of blood varied according to the study
site [Table 1]. The isolation efficacy was a uniform 77% when
46 out of 60 cultures were reported to be positive on each of the
human, locally produced or sheep blood agar when the SP was
in pure culture. This showed the laboratories missed 23% of
the culture even when the organism was in pure culture which
was misidentified. When the organism was with confounding
bacteria the isolation rates varied from 0% to 80% for the
isolation of SP from the mixtures.
Effects of transportation
To check for degradation of specimens, the second specimen in each
pair sent to each laboratory was returned undisturbed to the central
laboratory. The data loggers were checked for the maintenance of
temperature, and all shipments from and to the central laboratory
were within 2°C–6°C with no outliers. Each of the swabs was then
cultured on SBA, and for all organisms detected in the 1020 swabs,
there was uniformly no loss of the SP or the confounding bacteria
in mixture or pure cultures, in the swabs returned to the central
laboratory after double the time of transportation.
Study centre variability
There was variation in colony recognition among the study
centres. In the overall analysis, the different study centres
identified and processed varying amount of colonies with a
range of only 108 being processed from the 102 specimens
sent across by KA 01 centre, while MU 01 centre processed
317 colonies isolated on the various medium. The other
laboratories processed the isolates in varying numbers and
ranged from 125 to 140 colonies per centre.
The isolation efficacy varied from 0% to 80% among the
various centres showing a spectrum of isolation efficacy. It
was also seen that Kol1 was the best centre isolating 56/63 SP
from all cultures. On the locally produced medium, it was seen
that the centres using commercial blood agar medium produced
from Biomerieux, Delta, EOS and Hi‑media performed well
and the locally produced media using sheep and human blood
gave varying results in the isolation efficacy.
It was also noted that there was no standardisation of the
Gram’s, colonial morphology and biochemical testing for
reporting, with each laboratory following its own interpretation
criteria. Another observation was the variation seen in the
optochin and bacitracin zone diameter in susceptibility testing.
It was also seen that the laboratories used different schemas
for identification and reporting.[21]
366 Indian Journal of Medical Microbiology  ¦  Volume 37  ¦  Issue 3  ¦  July-September 2019
Discussion
The clinical relevance of microbiological reports critically
depends on the collection of appropriate specimens at
the appropriate time, proper storage and transport to the
microbiology laboratory with the minimum possible delay.
Classically, the isolation of fastidious organisms such as SP,
Haemophilus influenzae and Neisseria meningitides was to plate
the specimen immediately or to keep the specimen at room
temperature, with cold storage and transportation being totally
not advocated.[2] This is especially true of respiratory specimen
cultures where the main burden is in the community, which
in turn puts a severe constraint on both the primary isolation
identification and typing by national reference laboratories. SP
epidemiological studies have been constrained by this important
deficiency where case identification and microbiological typing
has suffered due to inability of laboratories to identify and
exchange specimen and isolates for studies.[11]
The isolation efficacy of SP, across the laboratories, shows
that the organism is recovered in varying proportions in the
media, and the laboratory practices used to identify the same
is different and is not standardised.
This raises the possibility of the misrecognition of colonial
types on the various media and more importantly would
increase the pressure on the processing of specimens and would
make the laboratories either underestimate or overestimate the
colonial types. It was seen that most of laboratories identified
120–130 colonial types to process, while some had as low as
108 and some had 317 colonies being processed. This shows
that there is gross under‑ or overreporting of the colonial types,
making errors in processing and identification.[35]
Another important observation was the non‑rationalisation of
colonial types in many laboratories without standardisation
of nomenclature. It is of note to understand that the
colonial morphology is the first step in the recognition of
the pathogen as the biochemical testing and result‑based
dichotomous‑branched identification is done using this basis.
Another problem was the colonial lyses which were again not
standardised.
When the colonial types reported for the isolation of SP was
reported alone, it showed that the most productive medium was
the locally produced medium. A reason for this apparent better
performance is made on the grounds that the microbiologists
and laboratory technicians were better prepared to recognise
the colonial types on this medium. This also showed that the
KOL 01 was the best study centre with the centre isolating the
organism from 56 out of the possible 63 isolations, which is an
excellent rate. Many centres reported SP, where none existed in
the culture swab, dispatched based on misinterpreted colonial
morphology and biochemical test.
This again brings to the fore that training and proper quality
control is critical in the recognition of the organism by the
participating laboratories. Most of the laboratories performed
below par as each of them needed to report 63 isolates in
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Amarnath, et al.: Isolation and identification of S. pneumoniae after cross‑country transport
the various mediums and many of them did not pick up the
organism even though this was the only organism present on
the plate. It is of interest that in most of the places the human
and sheep blood agars performed similarly where there was
a distinct advantage in the isolation of the organism from the
locally used medium, especially when mixtures were present.
When the isolate was analysed based on the material sent out
as SP in pure culture, we saw that the isolation of the organism
was similar from human and sheep blood agar where 46 out of
the possible 60 isolates was obtained, and it was seen that the
organism was not recognised from 14 swabs each on human
and sheep blood agar. This loss of isolation was more marked
when the organism was sent out as scanty in proportion,
whereas the isolation was much better when the organism
was sent out as heavy in proportion, with moderate being
in‑between. This brings to the fore the inability of the study
centres to recognise the pathogen even when the organism
was in pure growth and the loss of the organism is nearly 24%
which is a significant loss. Again this underscores the need to
standardise the recognition and reporting of this pathogen.
In the local media, it was seen that the standardised medium
from commercial suppliers far outperformed the locally
produced media since medium standardisation most probably
played a significant role in allowing the initial growth of the
microorganism and its isolation.
When the confounding bacteria was S. pyogenes, the isolation
rate dipped to a little over  30% with the maximum loss
occurring in sheep blood agar which would give the wide zone
of haemolysis on this agar where the study centres reported
only 10 out of a possible 30 isolates on sheep blood agar. This
isolation rate was similar on HBA where 11 out of possible 30
isolates was reported by the study centres which again showed
that the medium employed was not allowing the recognition
of the pathogen SP in the presence of beta‑haemolysis which
may have confounded the laboratories. Again it was seen that
the local medium employed by the study centres could get a
better isolation rate of 20 out of a possible 30 organisms where
the maximum loss occurred on locally produced sheep and
HBAs. This again underscores that the medium used should
be standardised, and more importantly, we need to observe the
colonial morphology carefully before we disregard a colonial
type as not pathogenic in the examination and reading of plates
and picking and interpreting identification tests.
When the confounding bacteria was S. agalactiae, the loss of
SP isolation was the maximum with the organisms only being
picked up when the concentration of SP was heavy in 6 out
of a possible 30 specimens. When the SP was moderate, the
isolation rate was 4 out of 30 and only 1 out of 30 when the
organism was scanty. This again underscores the need to look
for the pathogen in the presence of beta‑lytic colonies, and it
was found that sheep blood agar was a little better than HBA,
and the media produced commercially performed better again
than locally produced media. It is of interest to note here that
the maximum loss was seen when this organism was present.
Indian Journal of Medical Microbiology  ¦  Volume 37  ¦  Issue 3  ¦  July-September 2019
When a non‑haemolytic organism like M. catarrhalis was
present, the isolation rate was 53 out of possible 50 organisms
with the isolation being picked up even when SP was scanty.
Here, it was the Sheep blood agar which performed the best
along with commercially produced medium with some locally
produced human and sheep blood agars performing well. It
is of interest to note that the organisms reported could be
due to the easy recognition of an alpha‑lytic colony with the
waxy M. catarrhalis colonies being distinct and apparent for
laboratorians to recognise the pathogen. This contrasts very
much with the isolation of the organism from pure culture
where a lesser number of isolates was obtained as compared
to the organisms sent out.
Here, we say that the rate of organism isolation was better
when the SP was scanty and moderate, while when the SP
was present in heavy amounts, and there was loss of isolation.
This was paradoxical.
When the confounding organism was S. epidermidis, the same
pattern of isolations as seen with the presence of M. catarrhalis
was observed. Here, again it was noted that in the presence of
a non‑haemolytic colony, the isolation was better than when
a beta‑lytic colony was seen.
In the presence of S. epidermidis, we got 45 isolations
out of a possible 60 which is good that is 75%. Here,
again the sheep blood agar performed well along with the
commercially produced sheep blood agars and the HBA
produced commercially came a close second and the local
medium again significantly did not support the organism. It is
of interest here to note that in this group, the isolation in the
scanty proportion of S. pneumoniae the loss was maximum,
again underscoring the ability of the study centers to pick up
the organism in all proportions; while the isolation rate in the
moderate concentration was marginally lower in the isolation
rate. Here, since two distinct varieties were present, it was
possible to pick up the organisms easily.
The ability of the laboratories to pick up the pathogen in the
presence of a confounding alpha‑lytic organism was significant,
since we got the maximum isolation rate of 65 isolations. Here,
it is to be noted that the laboratories overreported the isolation
of the SP since the S. mutans was reported as SP, since we got
65 reports of a possible 60 isolations.
This underscores the overreporting if we are not careful in
the interpretation of the Grams nature of the organism and the
preliminary identification by optochin. It is to be remarked here
that just going by colonial morphology coupled to the Grams
nature would overreport the isolation of this organism and the
need to run controlled optochin, and perhaps, inulin would
really augment the isolation and identification requirement.
The advent of automated systems has also brought to the fore
the optimal utilisation of automated identification systems,
which can give erroneous results if not tested appropriately.
Overall it was seen that many of the study centers reported
Enterococcus (even though no such organism was sent across),
367
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Amarnath, et al.: Isolation and identification of S. pneumoniae after cross‑country transport
though the study protocol clearly defined the organisms present
in the swab, laboratories identified species which were never
included in the protocol. In some instances, the reporting of
Aeromonas hydrophila, Kocuria and Candida species showed
that the interpretation of automated testing equipment and other
commercial systems may have been obtained due to mixtures
used or laboratory contamination. One limitation of the study
was that this study was on simulated specimen mixtures and
would need to be evaluated by transportation of actual patient
specimens.
Conclusion
The study clearly elucidates the ability of laboratories for
isolation of SP on HBA in resource‑limited settings. The
results highlight the difficulties inherent in correctly identifying
pathogens in mixed cultures; thus suggesting improvement
in using standardized tests across the study centers. The study
also reaffirms the ability to transport biological specimens over
long geographical distances without loss.
Acknowledgements
The authors would like to thank Ramanna L, Poornima L,
Daniel NK and Chamundeshwari K for their technical help.
The authors would also like to thank Pradeep KV and Deepa M
for data entry and Kunder P for data analysis of this work.
Heinrichs J, Antonello J, Walter S and Mathur G are also
thankfully acknowledged for their help in the coordination
with Merck Inc.
Financial support and sponsorship
Funding for this research was provided by MSD Pharmaceuticals
Pvt. Ltd. All authors were investigators for the sponsor. Data
from this manuscript were presented as a posted abstract
P‑055/ISPPD‑0344 conference at Hyderabad, India, in 2014.
Conflicts of interest
There are no conflicts of interest.
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