Degree project, 30 ECTS [January 13 2021]
Prevalence of ctrA and crgA genes in
non-meningococcal neisserial species colonising the
upper respiratory tract among university
students in Örebro
Version 2
Author: Magnus Klinteskog, Master in Medicine
School of Medical Sciences Örebro University Örebro Sweden
Supervisor[s]: Susanne Jacobsson, PhD
Örebro University hospital Örebro Sweden
Word count
Abstract: [242] Manuscript: [3786]
1 Abstract:
Introduction: A Neisseria meningitidis carrier study has been conducted among students at
Örebro university in Sweden in 2018 and 2019. Pharyngeal samples were collected from 3489 students. PCR for the genes ctrA and crgA was run on all samples. The positive samples were then cultured on agar plates to find the N. meningitidis. In 349 of the PCR positive samples, no N. meningitidis could be isolated, which raised the question if other bacteria could have these genes. The most likely bacteria to have these genes were assumed to be other species within the genus Neisseria.
Aim: To identify whether other neisserial species have the ctrA and crgA genes.
Methods: The 349 samples were cultured on agar plates for two days. The species were then
identified by MALDI-TOF MS. The isolated Neisserial species and some other species as controls were saved. PCR for ctrA and crgA genes were then run on these bacteria to determine whether they possessed these genes.
Results: Five N. meningitidis that had been missed by the first round of culture were
identified. Seventy-five other colonies of neisserial species were isolated. N. subflava (n=40) were the most common. Nine (12 %) were crgA positive but none were ctrA positive. At least one crgA positive colony was found in four of the five different non-meningococcal neisserial species isolated in this study.
Conclusion: The crgA gene seems quite common among non-meningococcal neisserial
species while ctrA seems to be specific for N. meningitidis.
2 Introduction
Neisseria meningitidis is a commensal bacterium that can cause life threatening meningitis and sepsis mostly in children younger than 4 years of age and among adolescents but also in the elderly e.g. with a more recent increase disease caused by serogroup Y N. meningitidis [1–5]. N. meningitidis spread by respiratory droplets and colonizes the upper respiratory tract [2,4,6,7]. Characteristic for N. meningitidis is that disease outbreaks occur as epidemics [2,8,9]. In the meningitidis belt in Sub-Saharan Africa, epidemics of serogroup A N.
meningitidis were common until the vaccination program against this serogroup was
conducted [10]. While epidemic outbreaks are more characteristic, sporadic cases does occur [2].
N. meningitidis is part of the Neisseria genus within the Neisseracae family. The Neisseria genus include several neisserial species of which only N. meningitidis and N. gonorrhoea are associated with disease [6]. Even if N. meningitidis can cause serious diseases it is more common to carry them in the nasopharynx while the carrier is asymptomatic [2–5,11]. Studies have shown that in the general population, the prevalence of N. meningitidis carriage is between 8% and 25% [12]. Risk factors for carriage include exposure for cigarette smoke, upper respiratory tract infections, being male as well as frequenting bars and parties [4]. Further risk factors are living closely with other young people, for example during military service [3], or attending gatherings like scouts camps [11]. However, the most important risk factor is age, as adolescents and young adults have a higher prevalence of carriage [4,12]. Serious disease is most likely to occur within 10-14 days after getting infected while asymptomatic carriage often continues for several months [2].
Asymptomatic carriers can spread the bacteria to other people which may get the disease, therefore it is important to know the prevalence of asymptomatic carriage of N. meningitidis in the population [2]. It is also important to determine which serogroups are the most
common, as the risk of disease is higher among some serogroups. The WHO recognizes twelve serogroups of which A, B, C, W135, X and Y are most likely to cause illness [12,13]. Several different vaccines have been developed to decrease the risk of N. meningitidis disease. The vaccination programs have shown various degrees of success as there is unclear evidence of effect of vaccination against serogroup B, A, C, W and Y in the United States while vaccination against serogroup C have almost eliminated serogroup C disease in Europe and Brazil [4,12]. As the effectiveness of the vaccine depend on the kind of vaccine
3 and what serogroup it is effective against, knowing the serogroup of the meningococci when doing carrier studies are important [4,8–10,14].
Among the Scandinavian countries, few carrier studies have been conducted. A study in 1994 in Oslo found that 9.6% in the randomly sampled study population carried N.
meningitidis [15]. An additional carrier study among 2296 12-24 years old in Norway with samples collected in 2018 and 2019 found that 7.3% were carriers [5]. After an outbreak of N. meningitidis disease in 2015 among scouts attending the World Scout Jamboree in Japan a study of the Swedish participants showed a 8% prevalence [11]. Recently a carrier study in Sweden has been initiated in Örebro to examine the prevalence of N. meningitidis carriage among university students. In this study, Polymerase-chain reaction (PCR) for the genes ctrA and crgA was used to identify samples with N. meningitidis [16]. The samples which were PCR positive were then cultured in order to isolate the N. meningitidis for further analysis using whole-genome sequencing. However, no N. meningitidis were successfully cultured from a large part of the samples which begs the question of the sensitivity of culture and if there were some cross-reaction with some other bacteria in the PCR. Septicaemia and meningitidis caused by N. meningitidis are rapidly progressing diseases and good diagnostic tools are vital for determining the diagnosis. Over the last couple of decades, PCR has become one of the most important tools to rapidly diagnose a various range of diseases [17]. Therefore, it is essential to investigate whether the method is reliable when it comes to diagnosing infections with N. meningitidis.
CtrA is a capsule to cell-surface gene used to identify N. meningitidis. However, it’s known that at least 16% of N. meningitidis lack this gene and a capsule [13,18]. In the genus Neisseria only N. meningitidis have a polysaccharide capsule and it is assumed that the capsule is a large part of why it can cause disease [6,7]. This may explain why ctrA is more common in meningococci isolated from people in which the bacteria has caused disease [6]. While N. meningitidis is the only species in the genus Neisseria to possess a capsule, studies have shown that some capsular genes exist within other neisserial species even though they lack a capsule [6,7]. This makes it possible that other species may still possess the ctrA gene. CrgA, a gene involved in adhesion, is also commonly used for identifying N. meningitidis but is known to have caused false positives from some other bacteria such as N. lactamica [19] and from some Haemophilus influenza [20]. Hence the PCR for crgA is considered less specific for N. meningitidis than ctrA.
4 Aim
As it is important to be able to rely on the PCR for diagnostics of such serious diseases, as the ones caused by N. meningitidis this study aims to examine the samples from the carrier study conducted in Örebro that were PCR positive but culture negative for N. meningitidis to find whether non-meningococcal neisserial species and specific other bacteria in the samples are PCR positive for ctrA and/or crgA.
Hypothesis
As PCR is more sensitive than culture, N. meningitidis existed in the samples that were PCR positive but not in a sufficient amount to be culture positive. PCR for ctrA and crgA is hence not going to be positive for other species within the Neisseria genus.
A secondary hypothesis is that if other bacteria are positive for PCR it is most likely going to be for the crgA gene and not for the ctrA gene since the ctrA gene is more specific to N. meningitidis.
Material and Method
Between September 2018 and September 2019, 3489 pharyngeal samples were collected from students at Örebro University as part of a carrier study to evaluate the prevalence of N. meningitidis carriage in the upper respiratory tract among asymptomatic young adults. The results of this study have not yet been published. Initially, N. meningitidis were detected by PCR targeting the N. meningitidis specific ctrA and crgA genes. If positive, the samples were subsequently cultured and preserved as part of the routine diagnostics. The samples were cultured on both selective and non-selective agar for one day in order to be more certain that as many N. meningitidis as possible were identified. The second day, the colonies which was suspected of possibly being N. meningitidis were separated and grown on non-selective agar for an additional day. On the third day, the species of the colony was determined by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). In 349 of the 601 PCR positive samples, the culture was negative for N. meningitidis and therefore included in this study (figure 1). Of these 349 samples, 49 were positive for both ctrA and crgA, 13 were only ctrA positive, and 287 were only crgA positive.
5
Figure 1: Schematic over inclusion criteria for the present study. Total number of samples collected in the
Meningococcal carriage study in Örebro and PCR positive/culture negative samples.
Because the upper respiratory tract is colonised by a wide variety of different bacteria several bacteria of interest were selected to study. These were primarily bacteria of the Neisseria genus as they are most likely to have a similar genome to N. meningitidis. However, it was also decided to include the three first isolates of H. influenzae and Moraxella catarrhalis to be discovered when cultured among these bacteria, as they are important pathogens and H. influenzae has previously displayed positivity for crgA and Moraxella is part of the Neisseraceae family. As a control, two isolates of Kingella
dentrificans were also included as they are from the Neisseraceae family and related to the Neisseria genus.
The study was then conducted in two steps. Culture and identification of the predetermined bacteria was the first step. Next step was to run ctrA and crgA PCR on these bacteria to determine whether they had ctrA and/or crgA genes.
Bacterial culture: In the first step the samples were taken from frozen stocks (-70 °C), re-cultured on selective chocolate agar which is used at Örebro university hospital for
identifying N. meningitidis containing colistin, vancomycin, trimethoprim and nystatin, at 37 °C in the presence of 5% CO 2overnight. This is the same selective agar used on the samples during the first culture. It was determined that non-selective agar was not needed on the first day because they tended to be overgrown with other types of bacteria than Neisseria and it would be difficult to separate these from the Neisserial species. N. Meningitidis has a specific look on agar plates, which was used by the researchers to separate from other bacteria even on non-selective agar plates, however other Neisserial species have more varied looks and are more difficult to separate from other species on non-selective agar. Hence, by using only selective agar on the first day and then separate possible Neisserial-species on the second day for culture on non-selective agar and not only possible N.
meningitidis, it was determined that enough non-meningococcal neisserial species would be identified for this study. Some non-meningococcal Neisseria would probably not grow on
3489 samples from the
upper respiratory tract 601 PCR positive
349 PCR positive but culture negative
These 349 were included for culture in this project
6 selective agar, but it was assumed that as many bacteria could be missed by not being able to identify and separate them on non-selective agar.
The second day, various colonies of interest were identified on the agar plates and
transferred to a new non-selective chocolate agar plate without antibiotics and incubated at 37 °C in 5% CO2 over an additional night. From these plates, species were identified by MALDI-TOF MS according to manufacturer's instructions (Burker Daltonics, Bremen, Germany) on the third day [11]. If any of the predetermined bacteria were identified by the MALDI-TOF MS, they were resuspended in 1 mL NaCl and frozen (-20 °C) until PCR could be conducted.
DNA extraction and PCR: The DNA from the collected bacteria was extracted using a custom protocol on the QIAsymphony platform (QIAGEN GmbH, Hilden, Germany) using the QIAsymphony DSP Virus/Pathogen Midi Kit, Version 1 (QIAGEN).
The PCR was run in 45 cycles to amplify the genetic material of the ctrA and crgA genes using MIC (Bio Molecular Systems, Upper Coomera, Australia), as previously described [16].The lower the Ct number the more genetic material was in the examined sample. Results
In the cultures 85 different colonies of the predetermined bacteria were isolated from a total of 79 (23% of the 349 examined) different samples. Of these 85, five (6%) were N.
meningitidis and were added to the isolates in the whole-genome sequencing group. The most common bacteria among the remaining 80 was N. subflava (n=40) (47%) (figure 2). Other neisserial species that were isolated was 20 N. lactamica (24%), ten N. flavescens (12%), three N. macacae (4%), and two N. mucosa (2%) (figure 2). Five M. catarrhalis were isolated and three (4%) were saved and included in the PCR. Among other bacteria in the neisseracae genus, only K. denitrificans were isolated. 67 colonies of K. denitrificans were identified from different samples, but only two colonies were saved and included in the PCR (figure 2). No H. influenzae were isolated.
7
Figure 2: Isolated bacterial colonies (n=85) from the predetermined bacteria of interest from the 349 PCR
positive/culture negative samples that were included in this study.
Of the saved 85 cultures, 80 had PCR-test for crgA and ctrA run on them after the 5 isolated N. meningitidis were removed to be included among the samples of N. meningitidis isolated previously in the carrier study. Zero (0%) were positive for ctrA. Of the 75 isolated meningococcal neisserial species, nine (12%) were crgA positive (table I). None of the non-neisserial species were positive for either ctrA or crgA (table II).
In table I we compare the crgA positive samples in this study with the PCR results from the original PCR run after the samples were taken in the carrier study. The amount of genetic material in each sample was slightly higher than in the first run, which was indicated by the fact that for most samples the PCR were positive at earlier cycles (Ct-value) than in the previous run. However, we find a relatively high number in most samples indicating a low amount of crgA in each sample (table I).
Table I: All ctrA and crgA positive samples from the PCR run conducted in this study compared to
the original PCR run conducted during the carrier study.
Species Ct-value* for ctrA Ct-value* for crgA Ct-value* for ctrA, original PCR Ct-value* for crgA, original PCR Difference in Ct-value* N. subflava 1 0 21 0 29 -8 N. subflava 2 0 22 0 39 -17 N. subflava 3 0 23 0 31 -8 N. flavescens 0 26 0 34 -8 N. mucosa 0 34 0 33 -1 N. subflava 4 0 35 0 28 -7 N. subflava 5 0 38 0 36 -2 N. subflava 6 0 38 0 29 -9 N. lactamica 0 24 0 37 -13 Average of N. subflava (6) 0 29.5 0 32 -2.5
Average of all positives 0 29 0 33 -4
*The lower the Ct-value, the more genetic material was present in the examined sample. 47% 24% 12% 6% 3%4%2%2% N. subflava (40) N. lactamica (20) N. flavescens (10) N . meningitidis (5) M. catarrhalis (3) N macacae (3) K. dentrificans (2) N. mucosa (2)
8 In table II, we see the result for how many isolates tested positive for crgA per Neisseria species isolated in the study. Only N. subflava had more than one positive colony with six (15%) positives. Highest percentage of positives were N. mucosa (50%) but with only one positive of two total isolates. None of the non-neisserial species were positive in PCR. Of the neisserial species, only N. macacae had no colony that were crgA positive, however there were only three colonies of N. macacae found in the study (table II).
Table II: Number of colonies with positive and negative PCR for crgA among each bacterial species
isolated with culture (n=80). PCR result (crgA) N. subflava (n=40) N. lactamica (n=20) N. flavescens (n=10) M. catarrhalis (n=3) N. macacae (n=3) K. dentrificans (n=2) N. mucosa (n=2) All (n=80) Negative 34 (85%) 19 (95%) 9 (90%) 3 (100%) 3 (100%) 2 (100%) 1 (50%) 71 (89%) Positive 6 (15%) 1 (5%) 1 (10%) 0 0 0 1 (50%) 9 (11%)
In table III we examine the original runs in the carrier study for the N. meningitidis (n=5) isolated here. These were not included in the PCR for this study as stated above so no new PCR was run on them. Of the five N. meningitidis found among the 349 samples included in this project only one was ctrA positive in the first round. All five were crgA positive in the first round, however the average of amplification cycles before detection was 35.39 indicating that only a small amount of genetic material existed in the sample (table III).
Table III: N. meningitidis isolates (n=5) identified in this study and ctrA/crgA positivity in the original
PCR run. Ct-value* for ctrA Ct-value* for crgA N. meningitidis 1 0 34.56 N. meningitidis 2 0 35.23 N. meningitidis 3 0 36.42 N. meningitidis 4 0 36.43 N. meningitidis 5 33.28 34.29
Average of the positive 33.28 35.39
*The lower the Ct-value the more genetic material was in the examined sample. Discussion
Relatively few isolates of most of the neisserial species were found. Among the 349 samples only 80 different neisserial colonies were found. Interestingly, N. subflava, which was the most common with about half of all Neisseria isolates, have a relatively high prevalence of the crgA gene with 15% positivity. None of the isolated non-meningococcal neisserial species were identified in a sample positive for ctrA in the original run except for one of the isolates of N. meningitidis which was not included in the PCR run for this project. Hence it
9 is not surprising that none of these bacteria were positive for ctrA in the second run with PCR.
In the study, the Ct-value is slightly higher for the crgA positive Neisseria than for the corresponding sample in the previous run of PCR. This is explained by the fact that the Neisseria have been selectively cultured and then collected before being run through the PCR. It is thus a measure of the fact that we have more of this crgA positive species in the sample than previous when the sample included many other kinds of bacteria. Now it is only, or almost only, one species in the sample and with a much higher concentration. The fact that the only ctrA positive neisserial species identified in this study was a N. meningitidis strengthen the hypothesis that there is genetic material from meningococci in these samples, but they were unable to find these when the samples were cultured. Several possible reasons could explain why it has not been possible to culture them. It could be that no living meningococci exist in the sample, only genetic material. A second reason is that it could be in such a small amount that it is hard to isolate N. meningitidis from the sample. A third possible reason is that more non-meningococcal Neisseria than was expected are sensitive to the antibiotics in the selective agar used on the first day of this study. It could therefore be interesting to do further studies with the samples that are ctrA positive to see whether N. meningitidis could be cultured from these samples. These findings support the assumption that crgA is a less specific gene as we assumed when we began this study. Another factor that is important to note is that the selection agar used for the first day of culture is designed for selective growth of N. meningitidis and not for all species of the genus Neisseria. In a study conducted in 1991, some N. polysaccharea were found to be sensitive to colistin [21]. This could explain why no colonies of N. polysaccharea were isolated in this study even though this Neisseria also seem to be more common in children than in adults [22]. An additional study have also shown that some non-pathogenic neisserial species are sensitive to colistin such as some groups of N. cinerea, N. flavescens, N. subflava biovars subflava, flava, and perflava, N. sicca and N. mucosa [23]. There is a lack of
research on non-pathogenic Neisseria and the effect of the antibiotics included in the selection agar is therefore uncertain on many of the species [7]. However, one could hypothesize that the neisserial species that have been successfully cultured on the selection agar are the ones most closely resembling N. meningitidis in their genetic structure, thus making them more likely to have the ctrA and crgA genes. It’s thus possible that the initial calculation described above that it was better to use only selective agar on the first day was
10 wrong as more neisserial species than expected could be sensitive to the antibiotics.
However, there would still be a problem with isolating the neisserial species on non-selective agar which would take a lot more time and also could be unsuccessful in finding more non-meningococcal Neisseria as other bacteria could take over the agar plate and crowd out the Neisseria which would then be impossible or at least difficult to isolate. With this study, only 14 of 349 PCR positive but culture negative samples have been
explained and only 9 by identifying crgA genes in non-meningococcal Neisseria. Hence this can only be an explanation for a small part of the PCR positive samples where no
meningococci have been found in the culture.
That no H. influenza was cultured in the study is not particularly surprising as the selection agar it was cultured on was made for neisserial species and included antibiotics to impede the growth of other bacteria. Both trimethoprim and colistin inhibit H. influenza [24–26]. M. catarrhalis are resistant to both trimethoprim and colistin which explains why five were isolated [24,27]. However, other studies show that some Moraxella are sensitive to colistin [23]. Including more colonies of these important pathogens could have been interesting but because that would include removing antibiotics it would have been more difficult to isolate the neisserial colonies. It also would demand a lot more resources as a much larger number of bacteria would be able to grow on the agar plates meaning that much more time would be spent separating, culturing and identifying these bacteria. Thus a selection agar was needed. The fact that H. influenza is inhibited by the antibiotics open the possibility that some of the samples with positive crgA were positive because there were H. influenza with crgA genes in the samples, as previous studies have shown that some H. influenza are crgA positive [20]. When cultivated on the selection agar these bacteria would then have been repressed by colistin and trimethoprim.
MALDI-TOF is a further possible problem in the analysis. For several colonies it has shown different species of Neisseria when colonies have been run multiple times. It is likely that the program is not particularly good at separating some of the neisserial species from each other. It seems good at separating N. meningitidis as well as N. lactamica but for some of the other colonies the MALDI-TOF has shown some problems with identifying exactly which neisserial species it is. In these cases, the colonies have been run through the machine multiple times to get a higher degree of certainty and if it still has not been able to provide an answer with sufficient certainty, the neisserial species that the machine have provided as
11 the most likely has been chosen as the most likely species. Ultimately it seems as the
MALDI-TOF seems to lean towards N. subflava in cases were the colony is run through MALDI-TOF multiple times but many other neisserial species have been shown as the most likely species at times. This provide a degree of uncertainty to how good MALDI-TOF is at determining the species of these other species of the genus Neisseria. It is possible it cannot recognize some neisserial species which might be why none of these bacteria have been found in the study. This might result from the fact that there is a lack of research on non-pathogenic neisserial species as there is not that much interest in non-non-pathogenic Neisseria and the MALDI-TOF has not been sufficiently tested in order to recognize some serogroups of these species with a high degree of certainty [7].
The study assumes that other neisserial species are the most likely to carry ctrA and crgA genes. However, as noted above, crgA is known to be present in some H. influenza making it possible that it also is present in other bacterial species. The study can hence not say that there is likely to be N. meningitidis in the crgA positive samples, however, it seems likely that there are N. meningitidis in the ctrA positive samples, as none of the samples isolated in this study nor any study that have been encountered when researching for this study have found ctrA genes in other bacteria than N. meningitidis.
It seems that the crgA and ctrA PCR are quite specific and that the prevalence of N.
meningitidis carriage is more common than the prevalence in culture would signal. Further studies with an additional PCR targeting other meningococcal specific genes sodC and porA [28] are underway in these samples. The results from this confirming PCR will then be compared to the original PCR results and if positive in both PCRs the sample will be designated as positive for N. meningitidis.
Conclusion
The primary hypothesis is proven false as crgA does exist among other Neisseria species however, it is infrequent and because of the small sample size impossible to determine exactly how prevalent it is.
However, the secondary hypothesis is correct that if there are positive PCR, they will be positive for crgA since it is considered less specific to N. meningitidis as ctrA does not seem to exist in other Neisseria species. Hence this supports that ctrA is more specific to N. meningitidis than crgA. It also finds that the crgA gene exists in several neisserial species besides N. lactamica.
12 It also supports the hypothesis that N. meningitidis exists in the samples, or at least genetic material, but in too small of an amount to be able to culture. Even when considering the Neisseria identified in this study, 335 of the original 349 PCR positive but culture negative samples still have no explanation for why they were positive.
References
1. Säll O, Stenmark B, Glimåker M, Jacobsson S, Mölling P, Olcén P, et al. Clinical presentation of invasive disease caused by Neisseria meningitidis serogroup Y in Sweden, 1995 to 2012. Epidemiol Infect. 2017/05/08 ed. 2017;145(10):2137–43. 2. Stephens DS. Biology and pathogenesis of the evolutionarily successful, obligate
human bacterium Neisseria meningitidis. Vaccine. 2009/05/23 ed. 2009 Jun 24;27 Suppl 2(Suppl 2):B71–7.
3. Jounio U, Saukkoriipi A, Bratcher HB, Bloigu A, Juvonen R, Silvennoinen-Kassinen S, et al. Genotypic and Phenotypic Characterization of Carriage and Invasive Disease Isolates of Neisseria meningitidis in Finland. J Clin Microbiol. 2012 Feb 1;50(2):264. 4. Santos-Neto JF, Ferreira VM, Feitosa CA, Martinez-Silveira MS, Campos LC.
Carriage prevalence of Neisseria meningitidis in the Americas in the 21st century: a systematic review. Braz J Infect Dis. 2019 Jul 1;23(4):254–67.
5. Watle SV, Caugant DA, Tunheim G, Bekkevold T, Laake I, Brynildsrud OB, et al. Meningococcal carriage in Norwegian teenagers: strain characterisation and assessment of risk factors. Epidemiol Infect. 2020 Mar 31;148:e80–e80.
6. Claus H, Maiden MCJ, Maag R, Frosch M, Vogel U. Many carried meningococci lack the genes required for capsule synthesis and transportThe GenBank accession number for the sequence of the cnl-1 allele is AJ308327. Vol. 148, Microbiology. Microbiology Society; 2002. p. 1813–9.
7. Clemence MEA, Maiden MCJ, Harrison OB. Characterization of capsule genes in non-pathogenic Neisseria species [Internet]. Vol. 4, Microbial Genomics. Microbiology Society; 2018. Available from:
https://www.microbiologyresearch.org/content/journal/mgen/10.1099/mgen.0.000208 8. Alderson MR, LaForce FM, Sobanjo-ter Meulen A, Hwang A, Preziosi M-P, Klugman
KP. Eliminating Meningococcal Epidemics From the African Meningitis Belt: The Case for Advanced Prevention and Control Using Next-Generation Meningococcal Conjugate Vaccines. J Infect Dis. 2019 Oct 31;220(Supplement_4):S274–8.
9. Chen WH, Neuzil KM, Boyce CR, Pasetti MF, Reymann MK, Martellet L, et al. Safety and immunogenicity of a pentavalent meningococcal conjugate vaccine containing serogroups A, C, Y, W, and X in healthy adults: a phase 1, single-centre, double-blind, randomised, controlled study. Lancet Infect Dis. 2018 Oct 1;18(10):1088–96.
10. Fernandez K, Lingani C, Aderinola OM, Goumbi K, Bicaba B, Edea ZA, et al. Meningococcal Meningitis Outbreaks in the African Meningitis Belt After
Meningococcal Serogroup A Conjugate Vaccine Introduction, 2011–2017. J Infect Dis. 2019 Oct 31;220(Supplement_4):S225–32.
13 11. Jacobsson S, Stenmark B, Hedberg ST, Mölling P, Fredlund H. Neisseria meningitidis
carriage in Swedish teenagers associated with the serogroup W outbreak at the World Scout Jamboree, Japan 2015. APMIS. 2018 Apr 1;126(4):337–41.
12. McIntyre PB, O’Brien KL, Greenwood B, van de Beek D. Effect of vaccines on bacterial meningitis worldwide. The Lancet. 2012 Nov 10;380(9854):1703–11. 13. Castillo D, Harcourt B, Hatcher C, Jackson M, Katz L, Mair R, et al. Laboratory
methods for the diagnosis of meningitis caused by neisseria meningitidis, streptococcus pneumoniae, and haemophilus influenza; WHO manual. 2nd ed. Centers for Disease Control and Prevention (U.S.), World Health Organization, editors. 2011 Dec 1; Available from: https://stacks.cdc.gov/view/cdc/11632
14. Yaesoubi R, Trotter C, Colijn C, Yaesoubi M, Colombini A, Resch S, et al. The cost-effectiveness of alternative vaccination strategies for polyvalent meningococcal vaccines in Burkina Faso: A transmission dynamic modeling study. PLOS Med. 2018 Jan 24;15(1):e1002495.
15. Caugant DA, Høiby EA, Magnus P, Scheel O, Hoel T, Bjune G, et al. Asymptomatic carriage of Neisseria meningitidis in a randomly sampled population. J Clin Microbiol. 1994 Feb;32(2):323–30.
16. Hedberg ST, Olcén P, Fredlund H, Mölling P. Real-time PCR detection of five prevalent bacteria causing acute meningitis. APMIS. 2009 Nov 1;117(11):856–60. 17. de Filippis I, de Andrade CF, Caldeira N, de Azevedo AC, de Almeida AE.
Comparison of PCR-based methods for the simultaneous detection of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae in clinical samples. Braz J Infect Dis. 2016 Jul 1;20(4):335–41.
18. Sadler F, Fox A, Neal K, Dawson M, Cartwright K, Borrow R. Genetic analysis of capsular status of meningococcal carrier isolates. Epidemiol Infect. 2003
Feb;130(1):59–70.
19. Rebelo MC, Boente RF, Matos J de A, Hofer CB, Barroso DE. Assessment of a two-step nucleic acid amplification assay for detection of Neisseria meningitidis followed by capsular genogrouping. Mem Inst Oswaldo Cruz. 2006;101:809–13.
20. Hou T, Du Q, Wang L, Zhou H, Xi Y, Chen Z, et al. Cross reactivity of Neisseria meningitidis crgA diagnostic PCR primers with nontypeable haemophilus influenzae. Clin Lab. 2014;60 9:1425–9.
21. Anand CM, Ashton F, Shaw H, Gordon R. Variability in growth of Neisseria polysaccharea on colistin-containing selective media for Neisseria spp. J Clin Microbiol. 1991 Nov;29(11):2434–7.
22. Cann KJ, Rogers TR. The phenotypic relationship of Neisseria polysaccharea to commensal and pathogenic Neisseria spp. Vol. 29, Journal of Medical Microbiology. Microbiology Society; 1989. p. 251–4.
23. Knapp JS. Historical perspectives and identification of Neisseria and related species. Clin Microbiol Rev. 1988 Oct;1(4):415–31.
14 24. Li J, Nation RL, Milne RW, Turnidge JD, Coulthard K. Evaluation of colistin as an
agent against multi-resistant Gram-negative bacteria. Int J Antimicrob Agents. 2005 Jan 1;25(1):11–25.
25. de Groot R, Chaffin DO, Kuehn M, Smith AL. Trimethoprim resistance in
Haemophilus influenzae is due to altered dihydrofolate reductase(s). Biochem J. 1991 Mar 15;274 ( Pt 3)(Pt 3):657–62.
26. May JR, Davies J. Resistance of Haemophilus influenzae to Trimethoprim. Br Med J. 1972 Aug 12;3(5823):376.
27. Winstanley TG, Spencer RC. Moraxella catarrhalis: antibiotic susceptibility with special reference to trimethoprim. J Antimicrob Chemother. 1986 Sep 1;18(3):425–6. 28. Diallo K, Coulibaly MD, Rebbetts LS, Harrison OB, Lucidarme J, Gamougam K, et al.
Development of a PCR algorithm to detect and characterize Neisseria meningitidis carriage isolates in the African meningitis belt. PloS One. 2018 Dec
15
Etisk reflektion
Specifikt för min studie har jag inte haft behov av så mycket reflektion över etikfrågor. Alla nasofarynx-prover inhämtades innan jag blev involverad i meningokock-bärarstudien. Ingen kodnyckel finns tillgänglig så jag kan inte binda enskilda prover till individer. Godkännande för meningokock-bärarstudien finns som skulle inkludera min studie men etikgodkännande krävs egentligen inte då jag endast arbetat med bakterier som inhämtats i studien.
Ett etiskt dilemma som jag skulle kunna tänka mig är att det i något av proverna upptäcks serogrupper av N. meningitidis som har hög risk för att orsaka sjukdom. I detta fall finns ingen möjlighet att söka upp personen som har lämnat provet. Ifall denna möjlighet fanns skulle det vara intressant att fundera på om det hade varit värt att bryta sekretessen för provgivaren för att ge antibiotika för att hindra ytterligare spridning av dessa serogrupper. Det finns trots allt en liten risk att denna person med en sådan serogrupp antingen kan drabbas själv av invasiv sjukdom men även att hen sprider denna vidare till andra personer i sin omkrets som kan drabbas av exempelvis meningit. Detta är en frågeställning som man kanske måste ställa sig innan man gör
bärarskapsstudier där man letar efter bakterier som kan orsaka sjukdom, är det värt att man har en kodnyckel så att man kan leta upp personer med dessa typer av bakterier för att man ska kunna förhindra sjukdom.
I detta fall så hade man nog inte behandlat personen även om man skulle känna till att personen bär på vad som bedöms som en riskabel serogrupp. Frågan är dock när det är värt att faktiskt försöka använda studier för att identifiera personer med bakterier som kan orsaka sjukdom och om det är värt att försöka behandla dessa personer. Är det etiskt riktigt att samla in information om att en person bär på en farlig bakterie utan att faktiskt spara informationen om vem det är så att man kan behandla honom och eventuellt hindra att hen sprider detta vidare till andra?
Jag diskuterade detta med min handledare som sa att det redan bedömts som så att
integritetskränkningen som det krävdes inte var värt det när man förväntade sig hitta så få, om någon, som skulle vara värd att få profylaktisk behandling.
16
Populärvetenskaplig sammanfattning
Prevalens av ctrA och crgA gener hos icke-meningokock Neisseria-arter i halsen. Meningokocker är en bakterie som kan orsaka allvarliga sjukdomar som sepsis (blodförgiftning) och meningit (hjärnhinneinflammation). Denna bakterie förekommer hos en del av befolkningen i deras hals utan att den orsakar sjukdom. Endast en del serogrupper av bakterien orsakar sjukdom och det är viktigt att känna till förekomsten av dessa bakterier. Därför har en bärarskapsstudie utförts i Örebro för att undersöka detta där prover från halsen på unga vuxna på Örebro universitet inhämtades.
För att kunna identifiera meningokocker i proverna används en polymeras-kedjereaktion (PCR) som identifierar gener som är specifika för olika bakterier. I studien används PCR för generna ctrA och crgA som anses specifika för meningokocker. Därefter odlas proverna ut för att hitta
meningokocker.
349 prover var PCR positiva men inga meningokocker hittas i odlingarna. Därför tror vi att crgA och ctrA eventuellt kan förekomma hos andra arter i Neisseria släktet. Vi har därför odlat ut dessa prover för andra Neisseria arter. 75 har identifierats och kontrolleras med PCR för ctrA och crgA. I 12% av de Neisseria vi hittade fanns crgA och i fyra av fem av de andra arterna av Neisseria vi hittade fanns åtminstone en koloni med crgA. Å andra sidan fanns ingen positiv för ctrA. Detta är intressant eftersom vi kan säga att ctrA positivitet är ett säkert tecken på att meningokocker finns i ett prov medan crgA positivitet kan finnas hos många andra arter. Detta är viktigt för att vi ska få veta hur bra dessa PCR-prov är för att identifiera Meningokocker i prover.
17
Cover letter
To the Editor-in-Chief of the Journal of Clinical Microbiology Örebro, Sweden, Dec 30 2020
Dear Dr. Alexander J. McAdams
I would like to submit an article entitled ‘Prevalence of ctrA and crgA genes among non-meningococcal neisserial species in the upper respiratory tract’.
For this article we have examined whether the ctrA and crgA genes, normally considered specific for N. meningitidis, exist among non-meningococcal Neisseria species in the oropharynx. The samples were collected for a N. meningitidis carriage study among university students in Örebro 2018 and 2019. The analysis is based on 349 samples in this study that were PCR positive for N. meningitidis with a PCR detecting ctrA and crgA but negative when cultured, no N. meningitidis could be isolated. Each of these samples were re-cultured and isolated neisserial species were collected and analysed by the ctrA and crgA PCR. The crgA gene was identified in at least one colony of four of five non-meningococcal neisserial species and in 12% of all Neisseria isolates. However, the ctrA gene was not identified in any other Neisseria species.
To our knowledge this is the only study where all isolated Neisseria species have been checked for these genes. The findings are important for validating already existing presumptions that the ctrA gene is highly specific for N. meningitidis while crgA is less specific.
This article has not been submitted to any other journal. Both me and my supervisor have read and approved the final text of the article.
Kind regards
Magnus Klinteskog MB Örebro University
