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 ~  Abstract
 ~ Introduction
 ~  Materials and Me...
 ~ Results
 ~ Discussion
 ~ Conclusion
 ~  References
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  Table of Contents  
Year : 2019  |  Volume : 37  |  Issue : 3  |  Page : 363-369

Cross-country transport and isolation and identification of Streptococcus pneumoniae by use of alternate sources of blood supplemented media among laboratories in India

1 Medical Director, Manipal Clinics, Manipal Hospital, Bengaluru, Karnataka, India
2 Consultant Microbiologist, Manipal Hospital, Bengaluru, Karnataka, India
3 Consultant Microbiologist, Golwilkar Metropolis Health Services, (I) Pvt. Ltd., Pune, Maharashtra, India
4 Sr. Consultant, Department of Microbiology, BL Kapoor Memorial Hospital, New Delhi, India
5 Consultant Microbiologist, Shanti Mukund Hospital, New Delhi, India
6 Consultant Microbiologist, Yashomati Hospital, Bengaluru, Karnataka, India
7 M.S. Ramaiah Medical College, Bengaluru, Karnataka, India
8 Consultant Microbiologist, West Bank Hospital, Howrah, West Bengal, India
9 St. John's Medical College Hospital, Bengaluru, Karnataka, India
10 SS Microbiology Laboratory, Thane, Maharashtra, India
11 Consultant Microbiologist, Sri Aurobindo Seva Kendra, Kolkata, West Bengal, India
12 Consultant Microbiologist, Pushpanjali Hospital, New Delhi, India

Date of Submission04-Aug-2019
Date of Decision04-Sep-2019
Date of Acceptance04-Nov-2019
Date of Web Publication05-Dec-2019

Correspondence Address:
Dr. Satish Kumar Amarnath
Manipal Clinics, The Annexe, 98/1 Rustom Bagh, HAL (Old) Airport Road, Bengaluru - 560 017, Karnataka
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ijmm.IJMM_19_82

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 ~ 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

How to cite this article:
Amarnath SK, Joshi S, Abhyankar MN, Adhikary R, Beena H B, Chugh T D, Gandhi K D, Hittinahalli V, Indumathi V A, Rajavari M, Muralidharan S, Rao S S, Roy I, Saini N. 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

How to cite this URL:
Amarnath SK, Joshi S, Abhyankar MN, Adhikary R, Beena H B, Chugh T D, Gandhi K D, Hittinahalli V, Indumathi V A, Rajavari M, Muralidharan S, Rao S S, Roy I, Saini N. 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 [serial online] 2019 [cited 2020 Oct 25];37:363-9. Available from:

 ~ Introduction Top

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],[3],[4],[5],[6],[7],[8],[9],[10],[11],[12],[13],[14],[15],[16],[17],[18],[19],[20],[21],[22],[23],[24],[25],[26],[27],[28],[29],[30],[31],[32],[33],[34],[35],[36],[37],[38],[39],[40],[41],[42],[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 alternative sources of blood are used in the preparation of blood 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 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],[30],[31],[32],[33],[34],[35],[36],[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 Top

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 More Details 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 laboratory utilised an agar plate of their selection as a comparator [Table 1].
Table 1: Blood agar sources used in the study

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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) [Table 2] and [Table 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.
Table 2: Number of cultures performed at individual study sites

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Table 3: Percent recovery of isolates from either pure or mixed culture swabs

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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],[4],[5],[6],[7],[10],[11],[12],[13],[14],[15],[17],[18],[19],[20],[23],[24],[25],[26],[27],[28],[38],[40],[41],[42],[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 were received the principal investigator decoded and analysed the results.


Data from the centres were pooled and analysed using Epi-info version 6.02 software (Centers for Disease Control and Prevention), Atlanta, USA.

 ~ Results Top

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]

 ~ Discussion Top

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 influenza e and  Neisseria More Details 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 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.

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), 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 Top

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.


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 KunderPfor 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.

 ~ References Top

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  [Table 1], [Table 2], [Table 3]


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2004 - Indian Journal of Medical Microbiology
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