Population
This study was in response to a request for surveillance of pneumococcal carriage in Central Australia during an outbreak of IPD. Central Australia has a geographical area of 830 000 square kilometres and a population of over 46 000, with a population of 28 000 in the town of Alice Springs. Alice Springs Hospital is the main hospital for Central Australia. The target population in this study was the residents of Central Australia.
Collection and participation criteria
During March 18–21, 2011, nasopharyngeal swabs (NPS) were collected from consenting participants presenting to or visiting the Alice Springs Hospital Emergency Department. Those meeting the following criteria were excluded: Triage 1 and 2 patients, (requiring urgent or immediate care); apparently under the influence of alcohol or other drugs; having special needs; in police custody; and very recent arrivals (tourists) into Central Australia. Signed informed consent was obtained for collection of a single nasopharyngeal swab and for access to individual pneumococcal vaccination data. Consent included options for testing swabs for pneumococcus alone, and/or laboratory storage for future ethically approved studies of respiratory pathogens. Place of residence and date of birth were also collected. The Central Australian Human Research Ethics Committee provided a waiver for this surveillance study as it was in response to a disease outbreak requiring a timely response.
Sample collection
Once consent was obtained, NPS were collected using rayon tipped swabs on flexible aluminium shafts. The quality of the sample was recorded as good (inserted into the nasopharynx for 5 seconds), fair (inserted briefly into the nasopharynx), poor (external nares) or sample collected with the swab after nose blowing into a tissue [18]. Nose blowing is not a standard sample collection method but offers an alternative specimen collection for children who refuse a nose swab and who have visible nasal discharge [18]. Swabs were immediately placed into 1ml of skim milk tryptone glucose glycerol broth (STGGB) [19] and frozen on dry ice. After each day of collection, frozen samples were transferred to a liquid nitrogen vapour shipper for storage and transport within 6 days to a −80°C freezer at the Menzies School of Health Research laboratory in Darwin NT, Australia.
Pneumococcal culture
Following WHO guidelines for the routine laboratory culture of pneumococcus [19], 10 μl of the 1 ml NPS in STGGB were cultured and grown to determine the presence of pneumococcus by colony morphology, optochin susceptibility, the Phadebact© pneumococcus test and positive Quellung omniserum reaction. At least two presumptive pneumococcal colonies were selected for serotyping, including morphologically distinct colonies. Pneumococcal serotyping of isolates was by Quellung method [19]. Antibiotic susceptibility followed the calibrated dichotomous susceptibility (CDS) method for oxacillin (1 μg), penicillin (0.5 μg), tetracycline (30 μg), sulfamethoxazole/trimethoprim (25 μg), erythromycin (5 μg), chloramphenicol (30 μg) and azithromycin (15 μg) [20]. Isolates non-susceptible to penicillin or azithromycin had minimum inhibitory concentration (MIC) determined using E-tests (BioMerieux, France). Using the Clinical and Laboratory Standards Institute (CLSI) guidelines, penicillin breakpoints for intermediate resistance and resistance were > 0.06 ― ≤ 2 μg/ml and >2 μg/ml respectively [21]. Azithromycin breakpoints for intermediate resistance and resistance were > 0.25 - ≤ 0.5 μg/ml and > 0.5 μg/ml respectively [21]. Presumptive non-capsular pneumococci (NCSpn) were identified as optochin sensitive, Phadebact© pneumococcus test negative, bile soluble and Quellung Omni serum negative.
DNA extraction from NPS samples
DNA was extracted from 100 μl aliquots of each NPS using a QIAamp 96 DNA blood kit (Qiagen, Germany) following enzymatic lysis as described previously [22]. The DNA was used in both the probe-based quantitative real-time PCR (qPCR) targeting lytA and the serotype 1 detection by real-time PCR as described below. For the qPCR 2 μl of sample was used in each reaction and for serotype 1 real-time PCR 1 μl of sample was used in each reaction.
Pneumococcal detection and estimation of load by qPCR
Probe-based qPCR targeting lytA was used for detection of pneumococcus and estimation of pneumococcal load in NPS as previously described using forward (5′-TCTTACGCAATCTAGCAGATGAAGC-3′) and reverse (5′-GTTGTTTGGTTGGTTATTCGTGC-3′) primers and probe (5′- [6-FAM]-TTTGCCGAAAACGCTTGATACAGGG- [TAMRA]-3′), with product size of 101 bp [22, 23]. All samples were run in duplicate in a Rotor-Gene 6000 real-time thermocycler (Corbett Research). Five pneumococcal DNA standards (reference strain ATCC49619) from 9 to 90,000 genome copies per reaction were included in each run along with multiple template controls of common respiratory pathogens. The limit of detection for this assay is 8.92 cells, equivalent to 4460 cells/ml. Successful qPCR assays had a standard curve correlation coefficient and efficiency of > 0.99 and > 0.86 respectively. All samples and standards were run in duplicate and required to amplify within 0.5 cycles. Any samples that did not fit the acceptability criteria were re-tested up to two times before being rejected and considered negative for pneumococcal DNA.
Serotype 1 detection real-time PCR
For detection of serotype 1 from NPS samples, serotype 1 primers described for multiplex PCR serotyping by Pai et al. (2006), using forward (5′-CTCTATAGAATGGAGTATATAAACTATGGTTA-3′) and reverse (5′-CCAAAGAAAATACTAACATTATCACAATATTGGC-3′) primers, with product size of 280 bp [24] were used in a SYBR-based (Bioline, London) real-time PCR. For the positive control a high concentration (8.92 × 104 copies/μl) and low concentration (8.92 copies/μl) of pneumococcal serotype 1 DNA was used. Cycling conditions were as follows; 50°C 2 mins, 95°C 10 mins, 45 cycles of 94°C for 30 s, 54°C for 30 s, 68°C for 60 s. A melt curve was generated for each reaction and any curve that deviated from the control melt curve was considered negative for serotype 1 DNA. Melt-curve analysis was done between 50-99°C, with 1°C each step with weight of 90 s pre-melt conditioning on first step and 5 s wait for each step thereafter. Both SYBR-based real-time and melt-curve analysis were done in a Rotor-Gene 6000 real-time thermocycler (Corbett Research).
Serotype 1 multilocus sequence typing (MLST)
A single serotype 1 isolate from each of the serotype 1 culture positive NPS was analysed by MLST using modified primers as per Marsh et al. [21]. PCR products were sequenced, using the forward and reverse primers from each PCR (Macrogen, South Korea). Analysis of results was done using DNA Star (DNA star, USA); final sequences were submitted to the MLST database (http://www.mlst.net).
Statistical analysis
All results were entered and analysis of results was done using STATA version 11.