Malaria Journal PCR-based methods for the detection of L1014 kdr mutation in Anopheles culicifacies sensu lato

Om P Singh
National Institute of Malaria Research
Sector 8Delhi-110077DwarkaIndia

Prerna Bali firstname.lastname@example.com
National Institute of Malaria Research
Sector 8Delhi-110077DwarkaIndia

Janet Hemingway email@example.com
Liverpool School of Tropical Medicine
L3 5QAPembroke Place, LiverpoolUK

Sarala K Subbarao email@example.com
Aditya P Dash
Tridibes Adak
National Institute of Malaria Research
Sector 8Delhi-110077DwarkaIndia

Malaria Journal PCR-based methods for the detection of L1014 kdr mutation in Anopheles culicifacies sensu lato
10.1186/1475-2875-8-154BioMed Central Page 1 of 8 (page number not for citation purposes) Methodology Address: 3 13/704, Eastend Apartment, Mayur Vihar, Phase-I Extension, Delhi-110096, India and 4 World Health Organization, Regional Office for South-East Asia, World Health House, Indraprastha Estate, Mahatma Gandhi Marg, New Delhi-110 002, India * Corresponding author
Background: Anopheles culicifacies s.l., a major malaria vector in India, has developed widespread resistance to DDT and is becoming resistant to pyrethroids-the only insecticide class recommended for the impregnation of bed nets. Knock-down resistance due to a point mutation in the voltage gated sodium channel at L1014 residue (kdr) is a common mechanism of resistance to DDT and pyrethroids. The selection of this resistance may pose a serious threat to the success of the pyrethroid-impregnated bed net programme. This study reports the presence of kdr mutation (L1014F) in a field population of An. culicifacies s.l. and three new PCR-based methods for kdr genotyping.Methods:The IIS4-IIS5 linker to IIS6 segments of the para type voltage gated sodium channel gene of DDT and pyrethroid resistant An. culicifacies s.l. population from the Surat district of India was sequenced. This revealed the presence of an A-to-T substitution at position 1014 leading to a leucine-phenylalanine mutation (L1014F) in a few individuals. Three molecular methods viz. Allele Specific PCR (AS-PCR), an Amplification Refractory Mutation System (ARMS) and Primer Introduced Restriction Analysis-PCR (PIRA-PCR) were developed and tested for kdr genotyping. The specificity of the three assays was validated following DNA sequencing of the samples genotyped.Results:The genotyping of this An. culicifacies s.l. population by the three PCR based assays provided consistent result and were in agreement with DNA sequencing result. A low frequency of the kdr allele mostly in heterozygous condition was observed in the resistant population. Frequencies of the different genotypes were in Hardy-Weinberg equilibrium.Conclusion:The Leu-Phe mutation, which generates the kdr phenotype in many insects, was detected in a pyrethroid and DDT resistant An. culicifacies s.l. population. Three PCR-based methods were developed for kdr genotyping. All the three assays were specific. The ARMS method was refractory to non-specific amplification in non-stringent amplification conditions. The PIRA-PCR assay is able to detect both the codons for the phenylalanine mutation at kdr locus, i.e., TTT and TTC, in a single assay, although the latter codon was not found in the population genotyped.

Background

Anopheles culicifacies s.l. is the main malaria vector in the Indian subcontinent, affecting mainly rural areas, and contributes 60-65% of malaria cases in India [1]. As this is an endophilic vector, indoor residual spraying (IRS) of DDT is the main strategy used for its control. This species is resistant to DDT in most parts of India. Although DDT is banned in many countries, the recent endorsement by World Health Organization for the use of DDT for IRS for malaria vector control [2] has renewed interest in this insecticide. Pyrethroids are the most commonly used insecticides for IRS and the only insecticide class recommended for impregnation of bed nets due to their relatively low mammalian toxicity and rapid knock down effect on insects. In India, the use of pyrethroids was initiated in 1990s to control malaria epidemics in areas where An. culicifacies s.l was resistant to DDT and malathion. Pyrethroid resistance in An. culicifacies s.l. was detected in Surat district of Gujarat state, western India, soon after its introduction in vector control programme [3].

DDT and pyrethroids are neurotoxins that act on the voltage-gated sodium channels by modifying their gating kinetics, resulting in the prolonged opening of individual channels leading to paralysis and death of the insect. One of the mechanisms of pyrethroid resistance in insects is referred to as knock-down resistance (kdr) caused by reduced target site sensitivity. The phenotype is commonly conferred by a single point mutation (L1014F/S/ H) in the IIS6 segment of voltage gated sodium channel [4,5]. Other mutations in different regions of the gene also confer knock-down resistance in some insects [4,6], but among anophelines this is the only locus where point mutations have been reported to date conferring resistance. Only two points mutations have been reported in anophelines at this locus-L1014F in Anopheles gambiae [7] (West African kdr), Anopheles arabiensis [8] and Anopheles stephensi [9], and L1014S in An. gambiae [10] (East African kdr).

The Leu-Phe mutation at the kdr locus in An. culicifacies s.l. was reported by Hoti et al [11] using an allele-specific PCR assay, whose external primers Agd1 and Agd2 were based on An. gambiae sequences. However, authors failed to amplify the DNA region of interest using the primers Agd1 and Agd2 (see Additional file 1) due to mismatching with template DNA. As the evident cross resistance between DDT and pyrethroids in this resistant strain strongly suggested a kdr type phenotype, an attempt was made to confirm the presence of kdr-based insecticide resistance in An. culicifacies s.l. by DNA sequencing and developing alternative high throughput methods for kdr genotyping. The part of the voltage gated sodium channel spanning IIS4-IIS5 linker to IIS6 segments of An. culicifacies species A, B and C was first sequenced which contains at least five res-idues where mutations have been reported in other insects, namely, Met 918 in the IIS4-IIS5 linker, Leu 925 , Thr 929 and Leu 932 in IIS5 and Leu 1014 in IIS6 [6]. Based on the sequences obtained, three PCR based assays for kdr genotyping were developed, i.e., Allele Specific-PCR (AS-PCR), Amplification Refractory Mutation System (ARMS) and Primer Introduced Restriction Analysis-PCR (PIRA-PCR). These assays were tested on a DDT/pyrethroid resistant An. culicifacies population from Surat district and validated through DNA sequencing. The ARMS and PIRA-PCR were developed as alternatives of AS-PCR, which is often alleged to be non-specific [12][13][14]. Additionally, the PIRA-PCR was developed as a method by which both the codons of Phe (TTT and TTC) at kdr locus can be detected by a single assay.

Methods

Study area

Anopheles culicifacies s.l were collected from the villages of Surat district of Gujarat state (21-22°N latitude and 73-74°E longitude) located within three Primary Health Centres (PHC), Amladam, Bareda and Dadheda, where a significant level of resistance to DDT and pyrethroids (7-13% and 60-69% mortalities recorded in adults against 4% DDT and 0.05% Deltamethrin, respectively, in WHO's standard insecticide susceptibility tests) have been reported [3]. This An. culicifacies s.l. population is comprised of species B and C [3]. Indoor resting mosquitoes were collected using hand aspirators between 0600 and 0800 AM. The mosquitoes were preserved in isopropanol and DNA from individual mosquitoes was isolated.

DNA sequencing

Initially a 1.45 kb PCR product encompassing IIS4-IIS5 linker to IIS6 segment of voltage gated sodium channel was amplified from individual mosquitoes using primers Kdr1F (5'-CTG AAT TTA CTC ATT TCC ATC A-3') and Kdr1R (5'-TGG TGC AGA CAA GGA TGA AG-3') in a reaction mixture (25 μl) which contained 1× Buffer, 1.5 mM of MgCl 2 , 200 μM of each dNTP, 1.0 μM of each primers and 1.0 unit of AmpliTaq Gold ® taq polymerase (Applied Biosystem). The conditions of PCR were: initial denaturation at 95°C for 5 min, followed by 35 cycles of 95°C for 30 S, 48°C for 60 S and 72°C for 120 S, and a final extension step of 72°C for 7 min. The PCR products were reamplified by semi-nested PCR using primers Kdr1F and KdrR (5'-CGA AAT TGG ACA AAA GCA AAG-3'). The PCR conditions were same as in that of primary PCR. The amplification was checked on 2% agarose gel and PCR product was purified using Millipore's Montage ® PCR filter units following vendor's protocol. The purified products were sequenced directly using BigDye Terminator V3.1 (Applied Biosystem). The primers used for sequencing were the same primer used for PCR amplifications, Kdr1F and KdrR. Other internal primers used for sequenc-ing were-Kdr2R (5'-TTG AAA GCC ACG TAC CAT AAC A-3'), Kdr3R (5'-CGG AAC ACA ATC ATG AAG GA-3') and Kdr2F (5'-GAC CAC GAT TTG CCA AGA TG-3') ( Figure 1). One specimen of each of An. culicifacies species A, B and C were sequenced using these primers. For subsequent sequencing, two PCR fragments were amplified separately using primer sets Kdr1F + Kdr2R and Kdr2F + Kdr1R due to presence of large intron (1 kb) close to the end of IIS5. Both the PCR reactions (25 μl) contained 1× Buffer, 1.5 mM of MgCl 2 , 200 μM of each dNTP, 1.0 μM of each primers and 0.625 unit of AmpliTaq Gold ® taq polymerase. The conditions of PCRs were: initial denaturation at 95°C for 5 min, followed by 35 cycles of 95°C for 30 S, 48°C for 45 S and 72°C for 60 S, and a final extension step of 72°C for 7 min. A total of 25 An. culicifacies s.l. samples from Surat were amplified using these primers, purified with Qiaquick ® PCR purification kit (Qiagen) and sequenced directly using BigDye Terminator. Two specimens were heterozygous for Leu (TTA) and Phe (TTT) at position1014 indicating the presence of a kdr-based mechanism in An. culicifacies s.l. No other amino acid substitution was detected.

cDNA sequencing

The total RNA of An. culicifacies species A, B and C was isolated using TRIzol Reagent (Invitrogen Corporation) according to the manufacturer's instructions. cDNA synthesis and RT-PCR were carried out using AccessQuick RT-PCR System (Promega Corporation). The first strand cDNA was synthesized by avian myoblastosis virus reverse transcriptase (AMV-RT) at 45°C for 45 min, followed by denaturation of RNA/DNA hybrid and inactivation of AMV-RT at 95°C for 2 min. Second strand cDNA synthesis and subsequent DNA amplification were done by Tfl DNA polymerase at the following conditions: 35 cycles of denaturation at 95°C for 30 sec, annealing at 45°C for 30 sec, and extension at 72°C for 1 min followed by a final extension of 5 min at 72°C. The primers used for RT-PCR were Kdr1F and Kdr1R. The product was re-amplified by seminested PCR using primers Kdr1F and KdrR, and thereafter sequencing was performed using BigDye Terminator v3.1.

The DNA sequences obtained from cDNA and genomic DNA analyses were deposited in GenBank (accession numbers: GQ268331-GQ268333 and GQ279245-GQ279247).

Schematic diagram showing locations of the primers used for sequencing and PCR based kdr genotyping methods

PCR based assays

Three molecular methods for kdr genotyping were developed based on sequences obtained in this study, viz., Allele Specific PCR (AS-PCR), Amplification Refractory Mutation System (ARMS) and Primer Introduced Restriction Analysis PCR (PIRA-PCR) (Figure 1). In all the PCRbased assays, unless otherwise stated, uniform concentrations of reagents, except primers, were taken, which were: 1× Buffer, 1.5 mM of MgCl 2 , 200 μM of each dNTP and 0.5 unit AmpliTaq Gold ® per 15 μl of reaction mixture. All the PCR reactions were carried out in a GeneAmp ® PCR System 9700 (Applied Biosystem) thermal cycler. The three assays were tested on field collected An. culicifacies s.l for kdr genotyping and results were validated following DNA sequencing.

Allele-specific-PCR (AS-PCR) analysis

The strategy for designing of AS-PCR was similar to Martinez et al [15] and Hoti et al [11], but sequences and location of primers were different and were based on An. culicifacies sequences obtained through this study. Two external primers flanking kdr locus, KdrF (forward, 5'-GGA CCA YGA TTT GCC AAG ATG-3') and KdrR (reverse, 5'-CGA AAT TGG ACA AAA GCA AAG-3'), and two allele specific primers KdrA (forward, 5'-TAC AGT AGT GAT AGG AAA TTT A-3') and KdrT (reverse, 5'-ACT GCT AGG TTA CTT ACG ACA-3') specific for Leu and Phe alleles, respectively, were designed. The predicted sizes of allelespecific bands in PCR product were 127 and 186 bp for Leu (S) and Phe (R) alleles, respectively. A common band of 271 bp was also predicted to be formed by external primers. The scoring of genotypes was made on the basis of presence/absence of 127 and 186 bp bands where presence of 127 bp band only was scored as SS, presence of 186 bp band only as RR, and presence of both the bands as SR. The primer concentrations in PCR reaction were 1.5 μM of KdrF, 0.75 μM of KdrR 3.0 μM of KdrA and 2.0 μM of KdrT. The conditions of PCR were: initial denaturation at 95°C for 5 min, followed by 35 cycles of 95°C for 30 S, 48°C for 45 S and 72°C for 60 S, and a final extension step of 72°C for 7 min. The amplified products were run on 2% agarose gel containing ethidium bromide and visualized under UV in gel documentation system (Figure 2).

Amplification refractory mutation system (ARMS)

The allele-specific primers in AS-PCR are based on single base primer-template mismatch and are prone to nonspecific amplification due to mispriming in non-stringent conditions. Amplification Refractory Mutation System is based on incorporation of additional single base mismatch in allele-specific primers near 3' of primer which provides tremendous specificity to SNP (single nucleotide polymorphism) primers [16]. For ARMS the external primers, KdrF and KdrR, were same as in AS-PCR and two allele specific primers, KdrA1 (reverse, 5'-GGG TTA CTG CTA GGT TAC TTA CGg CT -3') and KdrT1 (forward, 5'-CTG GCT ACA GTA GTG ATA GGA AAT cTT-3') specific to Leu (S) and Phe (R) alleles, respectively, were designed using software available at http://cedar.genet ics.soton.ac.uk/public_html/primer1.html [17] with incorporation of single base mismatch at third base position from 3' end terminus (deliberate mismatched bases in primers are indicated in small letters). The predicted size of a common band, the S-specific and the R-specific bands were 271, 191 and 132 bp respectively. The primer concentrations in the PCR reaction were 1.5 μM of KdrF, 0.6 μM KdrR, 0.6 μM of KdrT1 and 3.0 μM of KdrA1. The conditions of PCR were: initial denaturation at 95°C for 5 min, followed by 35 cycles each of 95°C for 30 S, 48°C for 45 S and 72°C for 60 S, and a final extension step of 72°C for 7 min. The amplified product was run on 2% agarose gel containing ethidium bromide and visualized under UV (Figure 3). To check the specificity of ARMS, the assay was also performed with increased concentration of MgCl 2 (up to 3.0 mM) and AmpliTaq ® Taq DNA Polymerase (up to 2.5 units per 15 μl reaction mixtures) at an annealing temperature of 45°C.

Allele-Specific PCR (AS-PCR) for kdr genotyping

Primer-introduced restriction analysis-PCR (PIRA-PCR)

PIRA-PCR is based on incorporation of a deliberate mismatch in one primer so that a new restriction enzyme recognition site is created in the PCR amplicon in the presence of a specific allele in the target DNA. A PIRA primer KdrP (forward) with sequence 5'-TCC TGG CTA CAG TAG TGA TAG GAA AaT T-3' was designed, where a T-to-A mismatch was incorporated at the 3 rd base position from 3' end terminus (shown in small letter in primer sequence). The terminal AAaTT-3' sequence of the PIRA primer (KdrP) creates a recognition site for the restriction enzyme ApoI or XapI, i.e., AAATTY, in the amplified PCR product with the primer KdrR (reverse) in the presence of the Phe codon (TTT or TTC) in the template DNA at the kdr locus. Hence the PIRA-PCR is capable of detecting both possible codons of phenylalanine (TTT or TTC). With the Leu (TTA) allele, no ApoI restriction site is created.

For PIRA-PCR analysis, a PCR product was amplified using primers KdrP and KdrR with final concentration of 1.0 and 0.5 μM, respectively. The conditions of PCR were: initial denaturation at 95°C for 5 min, followed by 35 cycles of 95°C for 30 S, 48°C for 30 S and 72°C for 45 S, and a final extension step of 72°C for 7 min. Five μL of PCR product was digested with 5 units of ApoI restriction enzyme (New England Biolabs Inc) with 1× buffer in a final volume of 20 μl and incubated at 50°C for 1 hour. The restriction products were run on 3.0% agarose gel containing ethidium bromide and visualized under UV illumination (Figure 4).

The predicted size of PCR product amplified with primers KdrP and KdrR is 134 base pairs, which when digested with ApoI or XapI, will be cleaved into 106 and 24 bp fragments (with four bases 5'-overhang) in the presence of Phe allele (TTT/TTC). No cleavage is expected to take place in presence of Leu (TTA) allele. The 134 and 106 bp fragments can be easily resolved on 3% agarose gel whereas 24 bp fragment is normally not expected to be visualized or difficult to discriminate from primer-dimer. Therefore, 134 bp band was scored as S and 106 bp as R.

Validation of PCR based assays

All the mosquito samples genotyped as RS (n = 18) and RR (n = 1) were sequenced alongside 23 SS in order to validate assays and to check for other mutations in addition to the Leu-Phe change. The primers used for PCR amplification and sequencing were KdrF and KdrR. The sequence data were aligned and analysed using Mega 4 alignment utilities [18].

Hardy-Weinberg equilibrium analysis

The Exact Test of Hardy-Weinberg equilibrium using a Markov chain on three genotypes, i.e., SS, RS and RR was done using software Arlequin ver 3.11 [19] Results DNA sequencing revealed the presence of a Leu (TTA) to Phe (TTT) mutation at residue L1014 in a total of 19 samples, 18 of which were heterozygote and one homozygous resistant individual. No other mutations were recorded, including L995H mutation reported in Iranian An. culicifacies s.l. [20].

Testing of AS-PCR and ARMS assays on An. culicifacies s.l. samples from Surat revealed presence of discrete bands of expected sizes, as visualized on agarose gel. With PIRA-PCR also all the expected bands were seen, except 24 bp fragment resulting from the cleavage of PCR product (134 bp). Therefore, the presence of 106 bp cleaved product was scored as R. A total of 186 samples were genotyped using AS-PCR, of which 167 were assigned as homozygous susceptible (SS), one as homozygous resistant (RR) and 18 as heterozygote (SR) based on the criteria mentioned in section 'Methods'. All of the RS and RR, and 80 of the SS samples, as genotyped by AS-PCR, were also assayed with ARMS and PIRA-PCR assays, with similar results to the AS-PCR.

The result of DNA sequencing of 42 samples genotyped by all the three assays (RR = 1, SR = 18, SS = 23) were in agreement with PCR based assays. Thus all three assays are specific and can be used for genotyping.

Primer Introduced Restriction Analysis PCR (PIRA-PCR) for kdr genotyping The AS-PCR, based on a single mismatch at the 3' end of the allele specific primer, was specific and no mispriming was observed. The ARMS was specific even with increased concentrations of MgCl 2 (3.0 mM) and taq polymerase (3 Units of AmpliTaq Gold per 25 μl reaction).

The observed and expected heterozygote frequencies were 0.09677 and 0.10202 respectively, which did not differ significantly (p = 0.41220) from Hardy-Weinberg equilibrium.

Discussion

Malaria control in India relies on control of the major vector An. culicifacies. Knock-down resistance in An. culicifacies may be a serious threat to the continued use of pyrethroids, which are currently the insecticide class of choice for control of multiple insecticide resistant vector population by IRS and the only insecticide class available for the impregnation of bed nets. The present study provides the first direct evidence of the presence of the L1014F kdr mutation in Indian An. culicifacies.

The point mutation at residue L1014 leading to Leu-to-Phe change in voltage gated sodium channel is a most common mechanism associated with knock-down resistance in insects against DDT and pyrethroids. Variant replacements of this residue, L1014H and L1014S, have also been reported [4]. In anophelines, only two mutations, L1014F and L1014S, are reported conferring resistance [7][8][9][10]. No mutation at other location has been reported in anophelines except a novel mutation, L995H, reported in a single specimen of An. culicifacies s.l. from Iran [20]. During this study, screening of limited numbers of An. culicifacies s.l. samples from Surat didn't revealed presence of L995H mutation. The implication of such mutation in DDT/pyrethroid resistance and frequency in resistant population is yet to be investigated.

For monitoring of kdr resistance in vector population, there is a need of simple and reliable methods. AS-PCR is a convenient and inexpensive method used for kdr detection. This assay format has widely been used with the malaria vector An. gambiae in Africa [7,10]. For An. culicifacies s.l., an AS-PCR was designed by Hoti et al [11] using the external primers Agd1 and Agd2 originally designed for An. gambiae. These primers do not work with An. culicifacies s.l. from Surat district (see Additional file 1) due to mismatch with template DNA which was revealed after sequencing presented in this study and, therefore, not reliable. Further, a significant departure from HW equilibrium, resulting from deficiency of heterozygotes, had been noticed by the authors, while re-examining genotyping data of kdr in An. culicifacies s.l. by Hoti et al [11], which may be due to discrepancy in genotyping result. This necessitated the development of culicifacies-specific molecular assays for kdr genotyping.

The AS-PCR designed for SNP detection is based on single base primer-template mismatch at 3' of the primers. These primers are generally specific but in some instances the mismatched base does not prevent the extension process [12,13]. Mispriming with the primer specific for susceptible (kds) allele has also been reported in An. gambiae [21], where the mismatched base is at penultimate position from the 3' terminus. To avoid such mispriming, an ARMS method was developed, where an additional deliberate single nucleotide mismatch is incorporated near the 3' end of the allele-specific primers, which increases the specificity of the ARMS primers [16].

PIRA-PCR is a very reliable method for SNP detection that reduces the possibility of genotyping error due to mispriming. The PIRA-PCR was initially used for genotyping of kdr in An. gambiae by Janeira et al [22], where the mismatch in the PIRA primer was incorporated at the penultimate 3' base in order to create a restriction site. In the PIRA primer designed here for An. culicifacies s.l., the mismatch was incorporated at the third base position from 3' end, which seems to be advantageous, being less refractory to amplification in comparison to those where mismatch is at penultimate base. One of the most significant advantages of current PIRA-PCR over previously described methods is its capability to detect both possible codons of phenylalanine, i.e., TTT and TTC, whereas other PCR based assays can detect only one codon. Although in the single mosquito population tested only the A-to-T substitution was encountered, which is the most common substitution in mosquitoes [5], the possibility an A-to-C substitution in other mosquitoes cannot be ruled out and has recently been seen in Culex quinquefasciatus where both substitutions (A-to-T/C) exist [23]. It is possible that the A-to-C substitution is being overlooked in other species. The PIRA-PCR strategy described here could be used for screening of Leu-Phe based kdr for other mosquito species with little modification in primer sequences. The PIRA-PCR is relatively costly compared to AS-PCR or ARMS, due to involvement of a restriction enzyme step, and it is also slightly more time consuming. The PIRA-PCR may be misleading in case of incomplete digestion of amplified product where RR samples may be scored as RS.

Recently several other molecular methods in addition to PIRA-PCR have been used for kdr genotyping as alternative of AS-PCR such as Heated Oligonucleotide Ligation Assays (HOLA) [24], Sequence-Specific Oligonucleotide Probes (SSOP) [25], Fluorescence Resonance Energy Transfer/Melt Curve Analysis (FRET/MCA) [26] and fluorescent based genotyping by capillary electrophoresis [27], but most of these methods are expensive or require more sophisticated instruments. PCR-based methods on the other hand are easy, inexpensive and can be performed in the most of the laboratories equipped with Thermal Cycler.

Figure 1
1Schematic diagram showing locations of the primers used for sequencing and PCR based kdr genotyping methods. A: sequencing; B: AS-PCR analysis; C: ARMS analysis; D: PIRA-PCR. Harpoon represents primers; solid and dotted horizontal lines represent exon and intron, respectively.

Figure 2
2Allele-Specific PCR (AS-PCR) for kdr genotyping. Lanes 1 & 10: 100 bp DNA ladder; lanes 2-4: SS; lane 5: RR; lanes 6-8: SR; lane 9: negative control Amplification Refractory Mutation System (ARMS) for kdr genotyping Figure 3 Amplification Refractory Mutation System (ARMS) for kdr genotyping. Lanes 1 & 8: 100 bp DNA ladder; lanes

Figure 4 Primer
4Introduced Restriction Analysis PCR (PIRA-PCR) for kdr genotyping. Lanes 1 & 9: 100 bp DNA ladder; lanes 2-4: SS; lane 5: RR; lanes 6-8: SR
(page number not for citation purposes)
& 3: SS; lane 4: RR; lanes 5 & 6: SR; lane 7: negative control
AcknowledgementsThis collaboration was funded by the Wellcome Trust. Authors are thankful to Mr Uday Kumar and Mr Shri Bhagwan for excellent technical assistance.ConclusionThe Leu-Phe mutation (L1014F) in the voltage gated sodium channel, which generates the kdr phenotype in many insects, was detected in a pyrethroid and DDT resistant An. culicifacies s.l. population from Surat, western India. Three PCR-based methods (AS-PCR, ARMS and PIRA-PCR) were developed for kdr genotyping based on DNA sequences obtained from An. culicifacies species A, B and C. All the three assays were specific. The ARMS method was refractory to non-specific amplification in non-stringent amplification conditions. The PIRA-PCR assay is able to detect both the codons for the phenylalanine mutation at kdr locus, i.e., TTT and TTC, in a single assay, although the latter codon was not found in the population genotyped.Competing interestsThe authors declare that they have no competing interests.Authors' contributionsOPS: Conceived strategies for sequencing and PCR-based assays, carried out DNA sequencing and analysis, data analysis and manuscript preparation.PB: kdr genotyping and DNA sequencing.JH: guided the study and contributed to the manuscript. SKS, APD and TA: contributed to the manuscript preparation.All authors provided critical reviews of the manuscript and approved the final version.Additional materialAdditional file 1Evaluation of An. gambiae-primers for the amplification of IIS6 segment of the voltage gated sodium channel from An. culicifacies (Surat population)
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BMC Infectious Diseases A randomised controlled trial of the effects of albendazole in pregnancy on maternal responses to mycobacterial antigens and infant responses to bacille Calmette-Guérin (BCG) immunisation [ISRCTN32849447]

Alison M Elliott
Uganda Virus Research Institute
P.O. Box 49EntebbeUganda

London School of Hygiene & Tropical Medicine
Keppel StreetWC1E 7HTLondonUK

Proscovia B Namujju
Uganda Virus Research Institute
P.O. Box 49EntebbeUganda

Patrice A Mawa
Uganda Virus Research Institute
P.O. Box 49EntebbeUganda

Maria A Quigley
National Perinatal Epidemiology Unit
Oxford University
Old Road CampusOX3 7LFHeadingtonOxfordUK

London School of Hygiene & Tropical Medicine
Keppel StreetWC1E 7HTLondonUK

Margaret Nampijja email@example.com
Uganda Virus Research Institute
P.O. Box 49EntebbeUganda

Peter M Nkurunziza firstname.lastname@example.com
Uganda Virus Research Institute
P.O. Box 49EntebbeUganda

John T Belisle
Department of Microbiology
Colorado State University
Fort Collins80523ColoradoUSA

Moses Muwanga email@example.com
Entebbe Hospital
P.O. Box 29EntebbeUganda

James Ag Whitworth
Uganda Virus Research Institute
P.O. Box 49EntebbeUganda

BMC Infectious Diseases A randomised controlled trial of the effects of albendazole in pregnancy on maternal responses to mycobacterial antigens and infant responses to bacille Calmette-Guérin (BCG) immunisation [ISRCTN32849447]
10.1186/1471-2334-5-115BioMed Central Page 1 of 8 (page number not for citation purposes) Research article * Corresponding author
Background: Maternal schistosomiasis and filariasis have been shown to influence infant responses to neonatal bacille Calmette-Guérin (BCG) immunisation but the effects of maternal hookworm, and of de-worming in pregnancy, are unknown.Methods:In Entebbe, Uganda, we conducted a randomised, double-blind, placebo-controlled trial of a single dose of 400 mg of albendazole in the second trimester of pregnancy. Neonates received BCG. Interferon-gamma (IFN-γ) and interleukin (IL)-5 responses to a mycobacterial antigen (crude culture filtrate proteins (CFP) of Mycobacterium tuberculosis) were measured in a whole blood assay. We analysed results for binary variables using χ 2 tests and logistic regression. We analysed continuous variables using Wilcoxon's tests.Results:Maternal hookworm was associated with reduced maternal IFN-γ responses to CFP (adjusted odds ratio for IFN-γ > median response: 0.14 (95% confidence interval 0.02-0.83, p = 0.021). Conversely, maternal hookworm was associated with subsequent increased IFN-γ responses in their one-year-old infants ; p = 0.013)). Maternal albendazole tended to reduce these effects.Conclusion:Untreated hookworm infection in pregnancy was associated with reduced maternal IFN-γ responses to mycobacterial antigens, but increased responses in their infants one year after BCG immunisation. The mechanisms of these effects, and their implications for protective immunity remain, to be determined.

Background

Bacille Calmette-Guérin (BCG) immunisation is relatively ineffective against tuberculosis in the tropics, where the incidence of tuberculosis is high [1]. Helminth infection induces T-helper-2 responses and production of immunoregulatory cytokines and molecules that modulate responses to both helminths and bystander antigens; Thelper-1 and cytotoxic responses, required for immunity to viruses and intracellular bacteria, may be impaired [2]. Recognition of these effects has promoted speculation that helminth infection contributes to the observed reduced efficacy of BCG, and increased susceptibility to tuberculosis, in tropical countries [3]. BCG is often administered at birth in developing countries, so the observation that sensitisation to maternal schistosomiasis or filariasis in utero was associated with reduced interferon (IFN)-γ and increased interleukin (IL)-5 responses to mycobacterial antigens at one year of age, following BCG immunisation at birth, is of particular interest [4]. Hookworm is more widespread than schistosomiasis or filariasis [5], and may also suppress IFN-γ responses, both to hookworm, and to mycobacterial antigens [6]. Routine anthelminthic treatment after the first trimester of pregnancy, targeting hookworm anaemia, is now advocated, despite theoretical risks of teratogenicity (seen in animal models but not in man) [7], as benefits are expected to outweigh risks. However, the impact of this policy on other aspects of maternal and infant health has not been thoroughly investigated [8]. We wished to determine whether maternal hookworm infection had similar effects on neonatal responses to BCG to those reported for schistosomiasis and filariasis, and whether any such effects were removed by treatment for hookworm during pregnancy.

Methods

Mothers in the second trimester of pregnancy were enrolled in a randomised, double-blind, placebo-controlled trial of albendazole treatment during pregnancy, at Entebbe Hospital, Uganda, between June and August 2002. The study was designed to examine effects of albendazole treatment in pregnancy on immunological and disease outcomes in infants. The design was revised when the World Health Organisation announced new recommendations for treatment of helminths in pregnancy [9]. This paper presents data for the preliminary group of mothers, enrolled before the change of protocol. Mothers were included if they were resident in the study area, planning to deliver in hospital and willing to know their HIV status. Mothers with haemoglobin below 8 g/dl were excluded and treated for hookworm and anaemia. Other exclusion criteria were abnormal pregnancy or history of adverse reaction to anthelminthic drugs. Before treatment, mothers were screened for intestinal parasites by the Kato-Katz method and stool culture [10]; blood was examined for malaria, microfilariae [11], full blood count and HIV (within a programme for prevention of mother-to-child HIV transmission). All eligible mothers were then randomised to treatment with single-dose albendazole (400 mg) or placebo, regardless of whether hookworm was detected or not. The randomisation sequence was generated by an independent statistician (MQ), using blocks of 50. Albendazole and matching placebo tablets (Glaxo-SmithKline, UK) were packaged in identical envelopes, labelled with the randomisation code. Clinic staff gave participants the next number in the sequence, in order of enrolment.

At delivery mothers were asked to provide a second stool and blood sample; if possible, cord blood was obtained. Infants were immunised with intradermal BCG (100,000-330,000 colony forming units; Serum Institute of India, India). Six weeks after delivery all mothers were treated with both albendazole and praziquantel. Stool and blood samples were obtained from infants at one year of age.

All participants gave written informed consent. Ethical approval was given by ethics committees of the Uganda Virus Research Institute, Uganda National Council for Science and Technology and London School of Hygiene and Tropical Medicine.

Immunological assays

The primary outcome measures were immune responses in mothers and their infants, measured as follows. We examined stimulated IFN-γ and IL-5 responses in a whole blood assay, and measured serum levels of regulatory cytokines (IL-10 and Transforming Growth Factor (TGF)β). In the whole blood assay [12], unseparated, heparinised blood was diluted to a final concentration of one-in-four using RPMI supplemented with penicillin, streptomycin and glutamine, plated in 96-well plates, and stimulated with crude culture filtrate protein of M. tuberculosis (CFP) (5 µg/ml), or phytohaemagglutinin (PHA) (10 µg/ml) (Sigma, UK), or left unstimulated. Supernatants were harvested on day 6 and frozen until analysed.

Cytokine concentrations in serum or supernatants were measured by ELISA (Becton Dickinson, UK). The sensitivity of the assays was 62.5 pg/ml for TGF-β; 2.5 pg/ml for IL-10; 7.8 pg/ml for IL-5; 9.4 pg/ml for mothers and 37.5 pg/ml for infants for IFN-γ. Low-level cytokine production in un-stimulated wells was subtracted from concentrations produced in response to stimulation. Assays for samples with results above the highest standard were repeated after dilution.

Study profile

Statistical methods

Cytokine levels and responses were skewed so quantitative data were analysed using Wilcoxon's tests for paired or unpaired data as appropriate. A high proportion of mothers and infants showed positive IFN-γ responses to CFP. To allow analysis of effects of potential confounding factors using logistic regression, a binary variable was created indicating IFN-γ production below or above the median response. The sample size for this analysis was determined by the number of mothers recruited before the change in study protocol. Our analysis was not typical of a controlled trial, because our objective was to study both the effects of maternal helminths (by comparison of outcomes between mothers with and without helminths) and the modification of their effects by treatment (in the trial). Therefore, we first examined the effect of maternal helminth infection on cytokine responses in mothers at enrolment, and in their infants when they reached one year of age, and used the binary variable for high IFN-γ response to perform an analysis that adjusted for potential confounding factors using logistic regression. We then analysed the treatment trial by intention-to-treat: for each analysis, comparisons were made between albendazole and placebo recipients, first, for all participants, and second, for the sub-group of mothers with hookworm at enrolment (in whom any effect of treatment was expected to be greater). We examined the effect of treatment on responses in the mothers (using samples taken at delivery); in cord blood; and in one-year-old infants. Unblinded analyses were conducted by MQ. All other staff and participants remain blinded to treatment allocation as follow up continues.

Results

The trial profile is shown in figure 1. Mothers in albendazole and placebo groups were similar in age, HIV, malaria and helminth prevalence, and baseline cytokine levels and responses. After delivery the prevalence of hookworm was 0/47 (0%) in the albendazole group and 14/42 (33%) in the placebo group; other helminths remained similar in the two groups. Among one-year-olds, 3/63 had hookworm (one whose mother received albendazole, two placebo), 1/63 had Trichuris (albendazole-mother) and 1/54 Mansonella (placebomother).

Effects of maternal hookworm on responses mycobacterial antigens in mothers during pregnancy and their one-year-old infants Figure 2 Effects of maternal hookworm on responses mycobacterial antigens in mothers during pregnancy and their one-year-old infants. Effects of maternal hookworm on IFN-γ responses to culture filtrate proteins of Mycobacterium tuberculosis in (a) mothers at enrolment during pregnancy (before receiving study treatment) and (b) their oneyear-old infants, who received BCG immunisation at birth. IFN-γ responses shown are net production: low-level cytokine production in un-stimulated wells was subtracted from concentrations produced in response to stimulation to give a measure of antigen-specific response. "No worms": no helminths detected in stool or blood sample from mother either in pregnancy or at delivery. "Hookworm": mother had hookworm in pregnancy. Boxes show median value, 25th and 75th centiles; whiskers show lower and upper adjacent values and dots indicate outside values. P values <0.10 (Wilcoxon's rank sum test) are shown.

Immunological findings

Kato-Katz stool results for mothers at enrolment and all cytokine results were available for 90/103 mothers at enrolment, 79/93 at delivery (72 with results at both time points), and 57/63 one-year-olds. Whole blood assay data were missing when assays were unsatisfactory, and samples insufficient for repeats. Analysis of cord blood samples was restricted to 48/52 samples where mixing with maternal blood was excluded using an alkaline denaturation test [13]; serum cytokine results were complete for these 48 samples. Whole blood assay data were complete for only 33 of these cord blood samples: only five had a positive IFN-γ response, and one a positive IL-5 to CFP; therefore cord blood whole blood assay results are not presented further here.

Effects of maternal helminth infection on cytokine responses in mothers at enrolment and in their one-yearold infants

At enrolment, all helminth species were associated with lower maternal IFN-γ responses to CFP. This effect was most marked, and statistically significant, for hookworm (median 10 (interquartile range 0, 162) pg/ml compared to 173 (4, 471) pg/ml, p = 0.048) (Figure 2(a)). Five mothers had a positive IL-5 response to CFP; all had helminth infections.

Among one-year-old infants, the opposite effect was seen: IFN-γ responses to CFP were higher, for all species of helminth, among infants of mothers who had helminth infections in pregnancy. Despite effective treatment of maternal hookworm in approximately half the cases, this effect was also strongest for hookworm (median IFN-γ production 737 (220, 1805) pg/ml for infants of mothers with hookworm; 75 (24, 228) pg/ml for infants of mothers without helminths, p = 0.020) (Figure 2(b)). Nine infants had positive IL-5 responses to CFP; 6 had mothers with helminth infection.

It was considered possible that the associations between maternal hookworm and responses to CFP in the mothers, or in their infants, might be explained by confounding factors such as co-infection with other pathogens (maternal malaria or HIV), maternal age, socioeconomic factors, or maternal BCG immunisation. We investigated this by creating binary variables for IFN-γ production below and above the median level of response in mothers and infants. This allowed us to examine associations between such factors and responses to CFP (Table) and to adjust for their effects using logistic regression. The observed associations between maternal hookworm and IFN-γ responses to CFP in mothers and their infants were not explained by any of the variables examined: odds ratios for the associations between maternal hookworm and high IFN-γ responses to CFP, adjusted for maternal age, HIV status, malaria parasitaemia, education, BCG scar and schistosomiasis were, for mothers at enrolment: 0.14 (95% confidence interval 0.02-0.83, p = 0.021); for infants at one year of age: 17.65 (95% CI: 1.20-258.66, p = 0.013).

Analysis of the trial: effects of maternal treatment with albendazole on cytokine responses to CFP

Mothers who received albendazole had an increase in IFN-γ response to CFP (median increase +26 (-7, +293) pg/ml), whereas placebo recipients did not (0 (-279, +73) pg/ml) (p = 0.019)). Among hookworm-infected mothers, corresponding increases were +55 (0, +386) pg/ml for albendazole, and 0 (-44, +81) pg/ml for placebo recipients (p = 0.055). Thus, treatment of hookworm in the mothers resulted in a profile of CFP responses that was similar to the "no helminth" group ( Figure 3a). No effects of albendazole were observed on maternal IL-5 responses.

Conversely, IFN-γ responses to CFP were lower in infants of hookworm-infected mothers who received albendazole than in those whose mothers received placebo ( Figure 3b) and IL-5 responses less frequent, although these effects were not statistically significant. The BCG scar diameter in one-year-old infants followed a similar pattern, being significantly smaller in infants of hookworm-infected mothers who received albendazole (2.0 (1.0, 3.0) mm) than in those whose mothers received placebo (3.5 (2.8, 4.0) mm; p = 0.026). BCG scar diameter showed a positive correlation with the IL-5 response to CFP (ρ = 0.32, p = 0.011) but not with the IFN-γ response.

Non-specific immune responses

In most analyses, maternal helminths and albendazole treatment showed no effect on PHA responses or serum cytokine levels. However, cord blood IL-10 was lower for albendazole than placebo recipients, and this effect was statistically significant among hookworm mothers, (median 3 (0, 5) pg/ml versus 12 (4, 18) pg/ml, p = 0.031). TGF-β was detected in cord blood only for three mothers, all placebo recipients, of whom two were hookworm-infected.

Discussion

We present results of a preliminary study, the principal limitations of which are small numbers and the wide confidence intervals of some results. However, interesting contrasts were observed with previous reports and, within the study, between mothers and infants. To our knowledge, this is the first report of the immunological effects of treating helminths in pregnancy and, as such, suggests further important avenues of research.

Our study was designed to examine the effects of active helminth infection through parasitological examination of stool and blood samples, and the effects of albendazole treatment in pregnancy. Examination of a single stool sample is less than 100% sensitive for helminth infection [14], so some infected mothers would be misclassified as uninfected. This would lead to underestimates of helminth effects; thus we expect that the results presented for comparisons between mothers with and without hookworm and their infants are conservative. This misclassification would not affect the validity of the results of the trial (effects of albendazole vs placebo regardless of helminth infection status); or of the sub-group analysis (albendazole vs placebo) in mothers who had positive initial stool results for hookworm.

The most striking effects were observed for maternal hookworm, which was associated with reduced IFN-γ responses to the mycobacterial antigen, CFP, in the mothers, and with increased IFN-γ responses, following BCG immunisation, in their infants. These effects were not explained by measured confounding factors. Confounding factors not measured in this study might include genetic factors, which affect susceptibility to helminth infections [5]. Thus adults found to be helminth-egg-positive might be immuno-genetically different from those found to be egg-negative and immuno-genetic traits transmitted from mother to infant might possibly explain the effects observed. For example, a trait associated with stronger type 1 immune responses might lead to increased susceptibility to hookworm in the mothers and stronger responses to BCG in their infants. However, this explanation seems unlikely: the effects observed were reduced by albendazole treatment in pregnancy suggesting that an effect of active helminth infection may be a more probable explanation of the results.

The suppressive effect observed in mothers was in keeping with the results of studies in laboratory animals indicating suppression of type 1 responses to mycobacteria in the presence of helminths [15][16][17] and reduced efficacy of BCG against challenge with virulent Mycobacterium tuberculosis in mice co-infected with Schistosoma mansoni [18]. In humans, studies addressing the effects of helminths on immune responses to mycobacteria (measured as tuberculin skin test responses, or as responses to mycobacterial antigens in vitro), have had variable results, perhaps related to choice of comparison groups, or differences in effect by species or intensity of helminth infection [19][20][21][22]. In this randomised, placebo-controlled trial, the increase in response among mothers who received albendazole supports a causal role of hookworm in suppression of the response. Studies in both laboratory animals [16,17] and humans [20,23,24], including a placebo-controlled trial of albendazole treatment prior to BCG immunisation among helminth-infected, Ethiopian adults [24], have indicated a suppressive effect of helminths on the Effects of maternal hookworm and albendazole treatment on responses mycobacterial antigens in mothers at delivery and their one-year-old infants Figure 3 Effects of maternal hookworm and albendazole treatment on responses mycobacterial antigens in mothers at delivery and their one-year-old infants.

Effects of maternal hookworm, and maternal treatment with albendazole during pregnancy, on IFN-γ responses to culture filtrate proteins of Mycobacterium tuberculosis in (a) mothers at delivery and (b) their one-year-old infants, who received BCG immunisation at birth. IFN-γ responses shown are net production: low-level cytokine production in un-stimulated wells was subtracted from concentrations produced in response to stimulation to give a measure of antigen-specific response. "No worms": no helminths detected in stool or blood sample from mother either in pregnancy or at delivery. "Hk, plac": mother had hookworm in pregnancy and received placebo. "Hk, alb": mother had hookworm in pregnancy and received albendazole.  p=0.020 c immune response elicited by BCG. The contrasting effect of prenatal exposure to maternal hookworm observed in our infants was particularly unexpected in view of the earlier report of a suppressive effect of prenatal exposure schistosomiasis and filariasis on the IFN-γ response following BCG at birth [4]. One possible explanation is that immunological effects differ between helminth species. On the other hand, the earlier study of prenatal exposure did not include uninfected mothers, but compared infants sensitised, or not sensitised to helminth antigens in utero: suppression of IFN-γ responses to mycobacterial antigens following BCG immunisation was observed in the sensitised infants. Thus the nature of the exposure in utero, perhaps related to the species or intensity of helminth infection, may be an important factor.

Our results for IFN-γ could mean that light-to-moderate maternal hookworm infection induced a "better" response to BCG. However, noting that IL-5 responses were more frequent, and BCG scars largest, in the untreated hookworm group, and that scar size correlated with IL-5 production, an alternative hypothesis might be considered. Immunological factors in neonates, such as the elevation of regulatory cytokines suggested by our results for cord blood, may lead to delayed control of BCG replication, a larger "dose" of mycobacteria, and less selective, more destructive cytokine responses, which may, or may not, be protective [25][26][27]. This hypothesis will be investigated in further studies.

Conclusion

Maternal helminths, especially hookworm, were associated with suppression of maternal responses to the mycobacterial antigen, CFP, and this effect was largely removed by maternal treatment with albendazole. These results, and the elevated levels of IL-10 in cord blood of mothers with untreated hookworm, were in keeping with current hypotheses regarding the immunoregulatory effects of helminth infections.

By contrast, the association between maternal hookworm and an elevated IFN-γ response to CFP in infants one year after BCG immunisation was unexpected. The mechanism of this effect, and its implications for protective immunity, need to be explored.

Competing interests

The author(s) declare that they have no competing interests.

Authors' contributions

AME designed and ran the study, conducted data analyses and wrote the report. PBN and PAM conducted the immunological assays. MN contributed to care of participants. MAQ contributed to study design, statistical analysis, and

Figure 1
1Study profile.

Assessed

Table 1 :
1Factors associated with high IFN-γ responses to mycobacterial antigens among mothers and their infants.Mothers during pregnancy n = 90Infants at one year of age n = 57 The median level of IFN-γ production in response to CFP for mothers was 69 pg/ml; for infants 206 pg/ml. mv: missing values writing of the report; she conducted the unblinded analysis of the effect of albendazole: all other study staff have remained blinded to treatment status as the study continues. PMN conducted parasitological investigations. MM and JAGW contributed to study design, organisation, interpretation of data and writing of the report.Maternal variables in
pregnancy

Proportion (%) with "high IFN-
γ" (> median a )

P value
Proportion (%) with "high IFN-
γ" (> median a )

P value

Helminths
none
12/19 (63%)
4/17 (24%)
hookworm
11/36 (31%)
0.025
16/20 (80%)
0.001
S. mansoni
12/22 (55%)
0.75
7/12 (58%)
0.12
HIV infection
Negative
39/78 (50%)
26/49 (53%)
positive
5/12 (42%)
0.76
3/8 (38%)
0.47
Malaria parasitaemia
Negative
41/77 (53%)
25/49 (51%)
Positive
3/13 (23%)
0.07
4/8 (50%)
1.00
Age
< 25 years
28/61 (46%)
19/37 (51%)
25 or older
16/29 (55%)
0.50
10/20 (50%)
1.00
Education (1 mv)
none/ primary
29/55 (53%)
19/34 (56%)
secondary/ tertiary
15/34 (44%)
0.52
10/23 (43%)
0.42
BCG scar (3 mv)
absent
11/28 (39%)
10/14 (71%)
present
32/59 (54%)
0.25
18/41 (44%)
0.12

a
(page number not for citation purposes)
AcknowledgementsWe thank all participants and the staff of the "Mother and Baby (MAB) study": "The impact of helminths on the response to immunisation and on susceptibility to infectious diseases in childhood in Uganda". We thank especially the midwives of the Entebbe Hospital Maternity Department, the community field team in Entebbe and the staff of the microbiology laboratory of the Medical Research Council Programme on AIDS in Uganda.The study was funded by a Wellcome Trust Fellowship to AME, grant number 064693; albendazole and matching placebo were provided by Glax-oSmithKline; mycobacterial antigens were provided by JTB through the National Institutes of Health contract NO1-A1-25147; PMN and JAGW were supported by the British Medical Research Council Programme on AIDS in Uganda, grant number E743.The funding agencies had no role in the design and conduct of the study, collection, management, analysis and interpretation of the data; or in the preparation of the manuscript or decision to submit for publication. Glax-oSmithKline reviewed and approved the manuscript.Pre-publication historyThe pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/5/115/pre pub
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Questioning the application of risk of bias tools in appraising evidence from natural experimental studies: critical reflections on Benton et al., IJBNPA 2016

David K Humphreys
Jenna Panter
David Ogilvie
Questioning the application of risk of bias tools in appraising evidence from natural experimental studies: critical reflections on Benton et al., IJBNPA 2016
10.1186/s12966-017-0500-4C O M M E N T A R Y Open AccessNatural experimentsBuilt environmentPhysical activityRisk of bias
We recently read the article by Benton et al. which reviewed risk of bias in natural experimental studies investigating the impact of the built environment on physical activity (Benton et al., 2016; Int J Behav Nutr Phys Act 13:107). As a technical exercise in assessing risk of bias to understand study quality, we found the results of this study both interesting and potentially useful. However, it prompted a number of concerns with the use of risk of bias tools for assessing the quality of evidence from studies exploiting natural experiments. As we discuss in this commentary, the rigid application of such tools could have adverse effects on the uptake and use of natural experiments in population health research and practice.

Background

We recently read an interesting article by Benton and colleagues published in IJBNPA [1]. The authors should be commended for adapting a new Cochrane Risk of Bias Tool-the ACROBAT-NRSI (or ROBINS-I)-for use in assessing the risk of bias when appraising natural experimental studies of the built environment [1,2]. We agree with the authors' calls to improve the rigour, execution and transparency of reporting in natural experimental studies [3][4][5]. Despite our admiration of this study, it prompts concerns about the application of rigid appraisal tools to studies exploiting natural experiments. As researchers with experience using natural experiments to generate policy relevant evidence, we are concerned that using tools like ACROBAT-NRSI without careful consideration of the practical difficulties in generating research evidence in certain areas of public health may serve to set the bar of methodological acceptability too high. This could lead to a widespread downgrading of evidence from natural experimental studies, further entrenching an existing 'evaluative bias' in favour of interventions that are easier to evaluate [6].

Research that exploits natural experiments is both opportunistic and pragmatic and investigators are often severely limited by practical circumstances. While the logic of natural experimental studies resembles the randomised controlled trial, we would argue that in practice they are quite different. Consequently, basic methodological features expected in prospectively designed trials (e.g. blinding or allocation concealment) might be impossible in studies exploiting natural experiments due to the researchers' lack of control over the intervention. Therefore great care must be taken to consider the current state of the evidence as well as the contextual challenges that may limit the application of certain design features. In areas of population health that lack a strong research base, even severely limited observational designs exploiting natural experiments may provide an important advance in knowledge. Furthermore, assessing risk of bias in a single publication may be a misleading way of interrogating the plurality of the evidence base from well-designed natural experimental studies that combine several complementary study designs, datasets and analyses using approaches such as pattern matching or triangulation [7][8][9]. In such cases, the risk of bias for the entire evaluation may be low, even if the risk of bias in some individual analyses is high [10]. In the following sections we revisit the basic principles and purpose of natural experiments and consider the relevance of the ACROBAT-NRSI as a tool for appraising quality.

The theory and practice of using natural experiments to generate public health evidence Natural experiments are, by definition, events that occur outside the control of the researcher. They are not "conducted" or "designed"; on the contrary, they are discovered [11]. Natural experiments are events that naturally assign units (i.e. people, groups, places) to a particular treatment, intervention or exposure. They can include natural disasters, sudden economic shocks, changes to local or national policies, or changes to the built environment. Often it is not the event that researchers are interested in, but the circumstances it may (or may not) generate. For example, certain changes to the built environment may increase access to greenspace, reduce traffic congestion, separate cyclists from motor vehicles or make neighbourhoods more walkable. Researchers capitalise on the opportunistic changes in these factors to test theories about the causes of health behaviours or other phenomena by designing observational or quasi-experimental studies around the natural experiment. Where a change in exposure to a putative causal factor occurs, this should result in measurable changes in certain health behaviours (e.g. physical activity) when comparing populations that are exposed and unexposed to the changes.

There is a difference in opinion over what may legitimately be considered a natural experiment [12]. Some argue that the term 'natural experiment' should be confined to those events that naturally assign units to an intervention at random (or 'as-if' random) [11,13]. However, othersincluding the Medical Research Council (MRC) guidance on natural experiments-take a much broader approach, stipulating only that studies capitalise on unplanned "variations in exposure" [14]. This latter approach has become more popular in population health research in recent years. It advocates a more flexible and inclusive approach to the study of events that result in natural variation in exposures, which might be evaluated using a wide range of methods.

In reality the practice of designing evaluative studies around natural experiments is fraught with challenges. If the natural assignment of units to an exposure occurs in ways that are non-random, studies may be subject to the same threats to validity as observational studies and may require complicated statistical controls for confounders [11]. In addition, many natural experiments will be evaluated retrospectively, severely limiting the opportunities to employ 'gold standard' design elements such as objectively-measured outcomes or the use of 'well matched' control groups-a point Benton et al. prioritise, but which is rarely straightforward [3]. The successful use of natural experiments often depends on co-operation with practitioners to identify opportunities, methodological creativity and a degree of good fortune. Given that the rigour of natural experimental studies can so often be determined by factors beyond the researchers' control, one has to question the extent to which a risk of bias tool-such as the one proposed by Benton et al.-distinguishes between design weaknesses on the one hand, and practical obstacles in the context in which researchers are working on the other.

The use of risk of bias tools for natural experimental studies

Benton et al. comment that "According to the principles of the ACROBAT-NRSI four fifths of included outcomes are 'too problematic to provide any useful evidence on the effects of interventions' and one fifth have 'some important problems'" (p. 11). We do not question the technical assessment of bias diagnosed by the ACROBAT-NRSI tool, but we question the interpretation of results. Rather than contextualising these findings within the realistic possibilities in the selected field, it uses a benchmark that reflects common expectations across a much broader field of study (the so called 'hierarchy of evidence'). This approach prompted Benton et al. to conclude that all studies are "too problematic" to draw evidence from, whereas an alternative interpretation might be that all studies contain risks of bias that are inherent in this complex and challenging area of research, yet we can be more confident about some studies than others. If the aim is to inform policy, a more pragmatic interpretation of the evidence (and its limitations) is required.

This observation is not limited to natural experiments. A similar study by Movsisyan and colleagues appraised a number of systematic reviews to examine the appropriateness of the Grading of Recommendations Assessment, Development and Evaluation (GRADE) tool for interpreting reviews of complex interventions [15,16]. The authors found that systematic reviews of complex interventions were more frequently categorised as of "very low" quality than those of more "simple" (e.g. pharmacological) interventions. No single review of a "complex intervention" was deemed to be of "high" quality under the GRADE criteria, despite the presence of studies considered to be the "best possible evidence" in their respective fields [15,17]. The authors concluded that GRADE, in its current format, may not be suited to the appraisal of such studies.

The approach proposed by Benton et al. has potentially serious implications. For example, it might severely downgrade innovative and influential studies like that of Merom et al., one of the first to evaluate the impact of changes to the built environment on physical activity [18]. This study incorporated a number of innovative features, including the creation of graded exposure comparison groups and longitudinal monitoring of bike traffic using counters. Not only did the authors measure both subjective and objective outcomes, they also tested mechanisms that might help to explain the outcomes and thereby support causal inference, such as awareness of the new cycle routes-a strength of the design not reflected in ACROBAT-NRSI. Although the rigour of this study was not comparable to that of a randomised controlled trial, it has undoubtedly been influential in prompting others to identify, evaluate, and develop similar natural experimental studies [4,5,19,20].

One might question how a number of landmark natural experimental studies might fare under the adapted ACROBAT-NRSI proposed by Benton et al. Take for example John Snow's study investigating the causes of death related to cholera in mid nineteenth century London [21]. The most compelling evidence initially provided by Snow comprised of little more than a cross tabulation of cholera death rates in homes serviced by two water companies, one of which had moved its source intake pipe upstream while the other continued to extract potentially contaminated water in central London. This sudden natural change in exposure enabled a test of Snow's theory that cholera was a waterborne rather than an airborne disease [11,22]. Viewed in the context of the evidence at that time, Snow's is a classic example of a study opportunistically exploiting a natural experiment using a limited and pragmatic analytical approach, which generated crucial evidence of great theoretical and practical importance where few studies previously existed. Paradoxically, if these initial findings had been appraised using ACROBAT-NRSI, Snow's analysis would likely have been classified as unreliable.

Conclusion

We are not against the use of risk of bias tools in general. They can be used as a way of understanding the limitations of the evidence and justifying a call for methodological improvement-which, to their credit, is what Benton et al. have done in this paper. But much greater care is required in making sense of such appraisals for natural experimental studies. A unique feature of natural experimental studies is that key factors central to the design are outside the control of researchers. A more pragmatic approach would consider the wealth (or scarcity) of existing evidence and acknowledge the practical obstacles researchers may face. This would be aided by greater effort in documenting successful opportunities to exploit natural experiments, and the characteristics that may facilitate success (e.g. strong relationships with political or administrative bodies to identify opportunities, understand processes of assignment to interventions, or provide access to data, etc.). Consolidating information on these and other factors would help researchers recognise the most promising opportunities to exploit natural experiments and improve the rigour of the resulting studies.
 Acknowledgements JP and DO are supported by the Medical Research Council (unit programme number MC_UU_12015/6).FundingNo funding was obtained for this manuscript.Availability of data and material Not applicable.Authors' contributions DKH, JP and DO jointly conceived of the idea. DKH drafted the manuscript and all authors contributed to editing and refining the final draft. All authors read and approved the final manuscript.Competing interestOne of us provided one of the anonymous peer reviews of the original manuscript for IJBNPA.Consent for publicationNot applicable.Ethics approval and consent to participate Not applicable.• We accept pre-submission inquiries • Our selector tool helps you to find the most relevant journal Submit your next manuscript to BioMed Central and we will help you at every step:
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