Use of FTA Cards To Transport Throat Swabs and Oral FluidSamples for Molecular Detection and Genotyping of Measlesand Rubella Viruses

Bettina Bankamp,a Carolyn Sein,b* Elisabeth Pukuta Simbu,c Raydel Anderson,a Emily Abernathy,a Min-Hsin Chen,a

Jean-Jacques Muyembe Tamfum,c Kathleen A. Wannemuehler,b Diane Waku-Kouomou,a Elena N. Lopareva,a

Joseph P. Icenogle,a Paul A. Rota,a James L. Goodsonb

aDivision of Viral Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USAbGlobal Immunization Division, Centers for Disease Control and Prevention, Atlanta, Georgia, USAcInstitut National de Recherche Biomédicale, Kinshasa, The Democratic Republic of the Congo

ABSTRACT The genetic characterization of measles viruses is an important tool formeasles surveillance. Reverse cold chain requirements for the transportation of sam-ples to reference laboratories are challenging in resource-limited settings. FTA cardsfacilitate the transport of virologic samples at ambient temperature as noninfectiousmaterial; however, the utility of FTA cards for the detection and genotyping of mea-sles virus from clinical samples has not been evaluated. Throat swabs (TS) and oralfluid (OF) samples were collected from suspected measles cases in the DemocraticRepublic of the Congo. Virus detection (reverse transcription-quantitative real-timePCR [RT-qPCR]) and genotyping (endpoint RT-PCR) were compared for samples from238 suspected cases; these samples were either transported using the reverse coldchain or at ambient temperature on FTA cards. Virus detection showed excellentpositive agreement for OF samples compared to TS (95.3%; confidence interval [CI],91.6 to 97.4), in contrast to 79.4% (CI, 73.5 to 84.3) for TS on FTA, and 85.5% (CI,80.2 to 89.6) for OF on FTA compared to OF samples. Genotyping results obtainedfor a subset of samples indicated that 77.3% of all TS and 71.0% of OF sampleswould produce genotype information compared to 41.6% of TS and 41.3% of OF onFTA cards. Similar results were found for 16 measles-negative samples that wereconfirmed as rubella cases. Measles genotype B3 and rubella genotype 2B were de-tected. FTA cards have limited utility for virologic surveillance of sporadic cases ofmeasles; however, they can be a useful tool for the expansion of virologic surveil-lance in countries where the reverse cold chain is not available.

KEYWORDS FTA cards, genotyping, measles virus, rubella virus, sample transport

In 2011, the member states of the World Health Organization (WHO) African Region(AFR) set a goal to eliminate measles by 2020 (1). A cornerstone of the WHO-

recommended elimination strategies is the implementation of high-quality case-basedmeasles surveillance, including the collection of adequate samples for laboratoryconfirmation and the identification of measles virus (MeV) genotypes (2). MeVs areassigned to one of 24 genotypes based on the phylogenetic analysis of the 450nucleotides coding for the carboxyl-terminal 150 amino acids of the nucleoprotein(N-450) (3). Samples for the genetic characterization of circulating measles virusesshould be collected from at least 80% of outbreaks (4). In countries with endemicmeasles, this information serves to identify the endemic genotype(s). As countriesprogress toward measles elimination, analysis of circulating MeVs plays an importantrole in the documentation of the interruption of endemic transmission and identifica-tion of the sources of imported cases (5).

Citation Bankamp B, Sein C, Pukuta Simbu E,Anderson R, Abernathy E, Chen M-H, MuyembeTamfum J-J, Wannemuehler KA, Waku-Kouomou D, Lopareva EN, Icenogle JP, Rota PA,Goodson JL. 2019. Use of FTA cards totransport throat swabs and oral fluid samplesfor molecular detection and genotyping ofmeasles and rubella viruses. J Clin Microbiol57:e00048-19. https://doi.org/10.1128/JCM.00048-19.

Editor Angela M. Caliendo, Rhode IslandHospital

Copyright © 2019 American Society forMicrobiology. All Rights Reserved.

Address correspondence to Bettina Bankamp,bbankamp@cdc.gov.

* Present address: Carolyn Sein, World HealthOrganization, Geneva, Switzerland.

Received 10 January 2019Returned for modification 21 January 2019Accepted 16 February 2019

Accepted manuscript posted online 27February 2019Published

VIROLOGY

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Despite the endemic circulation of MeV in many countries in the AFR, there is apaucity of information on MeV genotypes compared to that for other regions (6). Theobstacles to expanding genotyping in the AFR include the reverse cold chain require-ments for transportation to one of the three regional reference laboratories (7). CurrentAFR guidelines recommend the collection of throat swabs (TS), which are stored in viraltransport medium and should be transported to the laboratory at 4 to 8°C and storedat �70°C (2).

Alternative sample types and methods for storage and transport of virologic sam-ples outside the cold chain could greatly enhance measles and rubella surveillance inresource-limited settings (8). Flinders Technology Associates (FTA) cards consist of filterpaper impregnated with reagents that lyse cells, denature proteins, and immobilizenucleic acids in the fibers of the matrix (9). After transfer to FTA cards, samples can beshipped at ambient temperature as noninfectious material (10–12) and the cards havebeen used successfully to transport RNA viruses (13–15). FTA cards have already beenused by some laboratories for the transport of samples for the detection of MeV (16).However, there are no published studies that have evaluated the rates of case confir-mation by reverse transcription-quantitative real-time PCR (RT-qPCR) or the success ofgenotyping of MeV from samples stored on FTA cards compared to that for TS or oralfluid (OF) samples that were collected and transported using the reverse cold chain.

While TS are recommended for the collection of samples for measles moleculartesting, OF is an attractive alternative due to the ease of sample collection (17, 18). OFis a mixture of saliva and gingival crevicular fluid which is secreted between the gumand the teeth and which contains immunoglobulin M, immunoglobulin G, and viralnucleic acids (19–21). Cellular material present in saliva is also included in OF samples(22) and may contain measles and rubella viruses. Endpoint RT-PCR and nested PCRwere used to demonstrate that OF is a suitable sample type for the detection of MeVRNA (23, 24). OF may be stored and shipped at ambient temperature in countries withtemperate climates (25); however, transport in countries with higher temperaturesrequires refrigeration.

Measles and rubella remain endemic in the Democratic Republic of the Congo, withperiodic large measles outbreaks causing substantial childhood morbidity and mortal-ity (26–29). In 2012, the estimated coverage with one dose of measles vaccine was 73%(30), while rubella vaccination has not yet been included in routine vaccinations orvaccination campaigns. In 2014, we conducted a field study to compare the utility ofFTA cards for the transport of TS and OF samples for the molecular detection andgenotyping of MeV and rubella virus (RuV) in the Democratic Republic of the Congo.

MATERIALS AND METHODSSample size. A sample size of between 152 and 215 is required to estimate a binomial proportion

with a desired precision (score with continuity correction, power analysis and sample size [PASS] v14)assuming a P value of 85%, alpha value of 0.05, and desired precision between 5% and 6%.

Ethical approval. The study protocol was reviewed by the Associate Director for Science/LaboratoryScience (ADS/ADLS) at the Center for Global Health, Centers for Disease Control and Prevention (CDC).The ADS/ADLS determined that, since the samples (serum, TS, and OF) were being collected during thestandard measles case investigation, further review by the Institutional Review Board (IRB) of the CDCwas not required. An explanation of the study was provided to every caregiver and verbal consentsought prior to sample collection.

Sample collection. Personnel at the Institut National de Recherche Biomédicale (INRB) in theDemocratic Republic of the Congo were trained in sample collection for use of FTA cards and sent tooutbreak sites to train local health facility staff and supervise sample collection. Specimens werecollected prior to the implementation of measles mass vaccination campaigns using Sigma-Virocult viralcollection and transportation systems (Fisher Scientific, Hampton, NH, USA) and Oracol collection devices(MMD, Malvern, UK). Tubes with 1 ml sterile saline, disposable pipettes (Globe Scientific, Paramus, NJ,USA), Whatman indicating FTA cards (Sigma-Aldrich, St. Louis, MO, USA), transport pouches for FTA cards(Sigma-Aldrich), tongue depressors (VWR, Radnor, PA, USA), and labels were provided to the healthfacilities. Posters describing sample collection and processing were displayed in participating healthfacilities (see Fig. S1 in the supplemental material). Case investigation forms were filled out for eachsuspected case, and data entry was performed at the INRB. From each suspected case, two TS and twoOF samples were collected. The two TS samples were collected by holding the two applicators as one,and the two OF samples were collected, one from each side of the mouth. One TS and one OF samplewere stored and transported at 4 to 8°C according to current measles surveillance guidelines (2). The

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second TS sample was replaced in its container and shaken vigorously for 1 min to elute the sample fromthe swab. The second OF sample was replaced in its container after the addition of 1 ml of saline andpushed down 10 times to elute the sample from the sponge. Next, 250 �l of the viral transport mediumcontaining the TS eluate and 250 �l saline containing the OF eluate were transferred to one FTA cardeach using disposable pipettes. After drying for 1 h at ambient temperature, the two FTA cards wereplaced in a transport pouch. Pouches were transported in biohazard bags to the laboratory at INRB atambient temperature. At INRB, pouches with FTA cards were stored at 4°C with desiccant until shipmentto the CDC (Atlanta, GA). Sera were collected, stored, and transported according to current measlessurveillance guidelines (2). Sera were tested at INRB for the presence of measles IgM using the Enzygnostanti-measles virus IgM kit (Siemens, Erlangen, Germany). Measles IgM-negative sera were tested for thepresence of rubella IgM using the Enzygnost anti-rubella IgM kit (Siemens). TS samples were transferredto cryovials (VWR) and stored at �70°C until shipment to the CDC. OF samples were centrifuged at2,000 rpm for 5 min at 4°C to collect the liquid from the sponge, transferred to cryovials, and stored at�70°C until shipment to the CDC. OF and TS samples were shipped to the CDC Atlanta on dry ice.

RNA extraction, RT-qPCR, and genotyping. Office paper punches were used to cut five 6-mm disksfrom each FTA card. After punching each card, the punches were cleaned with 10% bleach and 80%ethanol. The five disks were transferred to a 1.5-ml centrifuge tube with disposable forceps. A modifiedversion of the QIAamp Viral RNA Minikit (Qiagen, Hilden, Germany) extraction procedure was used toextract RNA from five disks per FTA card. One hundred fifty microliters phosphate-buffered saline (FisherScientific) was added to each tube, followed by 600 �l AVL buffer with carrier RNA. The disks wereincubated for 10 min at room temperature, followed by the transfer of 700 �l of the liquid to a cleantube. The remaining steps of the procedure were performed according to the manufacturer’s recom-mendations. An RT-qPCR assay targeting the measles N gene was used to determine copy numbers ofN gene-specific RNA (31). Sample RNA quality was assessed by RT-qPCR for the human RNase P gene (31).Eight (3.2%) of the samples that were RT-qPCR negative for measles had to be excluded because ofinsufficient RNA quality or quantity. Samples from suspected cases that did not have detectable measlesRNA in any of the four samples and samples from suspected cases that were positive for RuV IgM weretested for the presence of rubella RNA using the CDC diagnostic rubella RT-qPCR assay. This assay useda primer and probe set located in a well-conserved region close to the 5= terminus of the rubella virusgenome and was duplexed to detect the RNase P cellular reference gene (unpublished data). Templatesfor sequencing and genotyping of MeV were generated as described previously (10). Templates forsequencing and genotyping of rubella virus were generated using a two-amplicon method or bynested-set amplification (32, 33). Sanger sequencing was performed on an ABI 3500 Genetic Analyzer.Phylogenetic analysis was performed using MEGA6 (rubella) or MEGA7 (measles) (34, 35). GenBankaccession numbers are listed in the trees.

Statistical analysis. Microsoft Excel 2016 was used for data entry and to construct the boxplot.Analysis was completed in R 3.4 (36). Due to the absence of a gold standard test that is both 100%sensitive and specific, we present percent positive agreement and percent negative agreement withcorresponding 95% Wilson (score) confidence intervals. A loess smoother, a locally weighted polynomialregression method, was plotted to compare RNA load between sample types by day since rash onsetusing a span value of 0.8. The gray bands in Fig. 1 represent the confidence intervals for the loess curveusing a t approximation for the standard error (37, 38). For the figure, we added 0.1 to the copy numbersand then transformed to the log10 scale. Time since rash onset was truncated at 25 days, and the pointswere jittered on the x axis to visualize points with identical values.

FIG 1 Comparison of MeV RNA loads between sample types by day since rash onset. MeV RNA was detected by RT-qPCR. Log-transformed copy numbers areplotted against days since onset. Gray shading provides a visual of the uncertainty of each line. The dots with log 10�1 copy numbers represent (multiple)negative samples. (A) FTS versus TS. (B) FOF versus OF. (C) OF versus TS.

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Accession number(s). Sequences were deposited in GenBank under accession numbers MH752133to MH752190 (measles) and MH654795 to MH654808 (rubella). These accession numbers are listedbehind the WHO names in the phylogenetic trees.

RESULTSSample collection and exclusion of samples. Between March 2014 and September

2014, TS and OF samples were collected from 270 suspected measles cases. Samplecollection took place at 38 collection sites in eight provinces in the Democratic Republicof the Congo. From each suspected case, two TS and two OF samples were collected.One each of the TS and OF samples were processed and transported according to theprotocol recommended by the WHO (2), which specifies storage and transportation at4 to 8°C. The second TS and OF samples were transferred to FTA cards at the collectionsite, followed by storage and transport at ambient temperature. These samples will bereferred to as FTS and FOF. Of 270 suspected cases, 16 were excluded due toincomplete sample sets, insufficient RNA quality or quantity, missing patient informa-tion, or because they were positive for both measles and rubella. Sixteen suspectedcases were excluded from the measles-specific analysis because they were positive forrubella IgM and confirmed as rubella cases. Samples from the remaining 238 patientswere analyzed to evaluate the utility of FTA cards to transport samples for the detectionof MeV RNA.

Detection of MeV RNA on FTA cards. Of the 238 suspected cases included in theanalysis, 185 (77.7%) were measles IgM positive, 8 (3.4%) were indeterminate, and 45(18.9%) were measles IgM negative, reflecting the large measles outbreak that occurredin the Democratic Republic of the Congo in 2014. All IgM-positive and -indeterminatecases had detectable RNA either in the TS or OF sample; 96% had RNA detected in both.Therefore, the IgM-indeterminate patients were grouped with the IgM-positive cases inthe following analyses. Of 112 (47.1%) suspected cases with samples collected 0 to3 days after rash onset, 24 (21.4%) were IgM negative, but 18 (75.0%) of these haddetectable MeV RNA in at least one sample. For all time points, 31 of 45 (69%)IgM-negative cases were positive by TS or OF, demonstrating the utility of molecularmethods for case confirmation. Eleven suspected cases were IgM negative and had nodetectable RNA in any sample. Samples from these 11 cases were collected 0 to 7 daysafter symptom onset. It is possible that these were not measles cases. Ninety-ninepercent of the TS and 97.4% of the OF samples from IgM-positive or -indeterminatepatients contained detectable MeV RNA (Table 1). The positive agreement was reducedto 79.8% and 85.5% for FTS and FOF, respectively, compared to IgM. The percentage ofsamples from IgM-negative suspected cases that did not contain detectable MeV RNAranged from 42.2% to 53.3%.

For both TS and OF, 89.9% of the 238 suspected cases contained detectable MeVRNA, compared to 73.5% of FTS and 79.8% of FOF (Table 1). Both TS and OF were RNApositive in 204 (85.7%) cases and negative in 14 (5.9%) suspected cases (Table 2),indicating good agreement between these sample types. Twenty samples (8.4%)produced discordant results, resulting in a positive agreement of the OF compared toTS of 95.3% and a negative agreement of 58.3%. However, when comparing TS and FTS(Table 3), the number of discordant samples increased to 49 (20.6%). This increase wasmostly due to a larger number of cases in which the TS was positive but the FTS was

TABLE 1 Measles case confirmation by PCR compared to detection of IgM

Sample type

RT-qPCR result

IgM-positive specimens (n � 193)a IgM-negative specimens (n � 45)Total no. positive(% of all 238 samples)No. positive Positive % agreement (95% CI) No. negative Negative % agreement (95% CI)

TS 191 99.0 (96.3–99.7) 22 48.9 (35.0–63.0) 214 (89.9)OF 188 97.4 (94.1–98.9) 19 42.2 (29.0–56.7) 214 (89.9)FTS 154 79.8 (73.6–84.9) 24 53.3 (39.1–67.1) 175 (73.5)FOF 165 85.5 (79.8–89.8) 20 44.4 (30.9–58.8) 190 (79.8)aIncluding IgM indeterminate cases.

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negative. Based on these data, the positive agreement between FTS and TS was 79.4%and the negative agreement was 79.2%. In the comparison of OF and FOF (Table 4), thenumber of discordant samples was 38 (16.0%), again due to the larger number ofOF-positive but FOF-negative cases, with a positive agreement of FOF compared to OFof 85.5% and a negative agreement of 70.8%.

For each collection day and for all sample types, the amount of MeV RNA found inthe samples varied over a wide range (Fig. 1). For the 24 cases whose samples werecollected more than 7 days after rash onset, only three TS and three OF samples wereRT-qPCR negative. While the FTS and FOF showed a similar broad distribution of RNAloads as the TS and OF samples, the median copy numbers per reaction were lower forevery time point. Median copy numbers per reaction for TS and OF were 2.75 � 105

(interquartile range, 7.70 � 106) and 2.81 � 105 (interquartile range, 6.26 � 106), re-spectively. For FTS and FOF, the median copy numbers were 3.45 � 103 (interquartilerange, 1.16 � 105) and 6.8 � 103 (interquartile range, 3.65 � 105), respectively, or 80-and 41-fold lower. When comparing RNA copy numbers, the volume of sample loadedon FTA cards and the fraction of the sample that is contained in the disks used for RNAextraction must be taken into account. Based on these considerations, the RNAextracted from FTA cards should have a 1.9-fold lower concentration than the corre-sponding TS or OF sample (data not shown). These results indicated that OF is assensitive a sample for molecular detection as TS and that detection of MeV RNA fromsamples transported on FTA cards is less sensitive than from TS or OF samples.

Utility of FTA cards for genotyping. The main advantage of using FTA cards forsample collection would be for genotyping when other methods for sample transportare unavailable. A subset of samples were selected for genotyping based on thelocation or time of the outbreak to obtain a more complete description of thedistribution of circulating MeV in the Democratic Republic of the Congo. Therefore,some samples were chosen despite having viral RNA copy numbers below the limit ofdetection (LOD) for the measles genotyping RT-PCR of 1 � 104 copies per reaction(Table 5) (10). Genotyping was successful for all TS and OF samples with copy numbersabove the LOD but only for 86% and 80% of the FTS and FOF, respectively, with copynumbers above the LOD. While it was possible to obtain genotype information fromsamples with viral RNA copy numbers below the LOD, the success rate was low for allfour sample types (Table 5). Using 104 copies as a cutoff for successful genotyping, anestimated 184 (77.3%) of all TS, 169 OF (71.0%), 99 FTS (41.6%), and 98 FOF (41.3%)samples would provide genotyping results. These numbers account for the reducedsuccess rate of genotyping from FTA cards and are likely slight underestimations,because suspected cases that could not be confirmed by either serology or RT-qPCRwere included in the totals. These results indicate that FTA cards are useful forgenotype analysis, albeit with a reduced likelihood of success compared to that for TSor OF samples.

TABLE 2 Comparison of TS and OF for measles case confirmation by RT-qPCR

OF PCR result

TS PCR result (no. [%])

Positive Negative

Positive 204 (85.7) 10 (4.2)Negative 10 (4.2) 14 (5.9)Agreement (% [90% CI]) 95.3 (91.6–97.4) 58.3 (38.8–75.5)

TABLE 3 Comparison of TS and FTS for measles case confirmation by RT-qPCR

FTS PCR result

TS PCR result (no. [%])

Positive Negative

Positive 170 (71.4) 5 (2.1)Negative 44 (18.5) 19 (8.0)Agreement (% [90% CI]) 79.4 (73.5–84.3) 79.2 (59.5–90.8)

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Genotyping results for measles. All 58 genotyped samples belonged to genotypeB3. The sequences can be divided into three separate clusters (Fig. 2). Forty-twosequences in cluster 1 were identical or very similar to the named strain, MVi/Harare.ZWE/38.09, with a maximum of four nucleotide differences. These sequenceswere found in five of eight provinces during epidemiologic weeks 12 to 36 of 2014,indicating a wide temporal and geographic distribution of this lineage in the Demo-cratic Republic of the Congo. Cluster 2 comprised 14 sequences from Kasai Oriental andKatanga Provinces, and the two sequences in cluster 3 were obtained from EquateurProvince. For seven cases, at least one sample from an FTA card and one TS or OFsample were genotyped. In all cases, the sequences were identical, demonstrating thereproducibility of the genotyping procedure when performed with RNA extracted fromFTA cards.

Rubella. Of 45 measles IgM-negative sera, 22 were positive for rubella IgM. After theexclusion of six cases for reasons described above, RNA samples from 16 rubella caseswere tested by RT-qPCR to detect RuV RNA. Thirteen TS and 11 OF samples testedpositive for RuV RNA compared to 8 FTS and 5 FOF. The amounts of RuV RNA variedsignificantly among the samples from these cases. Less RuV RNA was recovered fromOF, FTS, and FOF than from TS (Fig. 3). All cases that had at least one positive RT-qPCRresult had a positive RT-qPCR result for the TS. Genotyping was successful for 14 rubellacases, including one case that was excluded from the statistical analysis. Five casesoccurred in May 2014 in Kasai Occidental Province in the town of Dibaya, and six caseswere detected in July 2014 in Sud Kivu province in the town of Kabare, indicatingrubella outbreaks in these towns. All genotyped RuVs belonged to the same lineage ofgenotype 2B (2BL2c) (39) and grouped with RuV genotype 2B viruses collected in theDemocratic Republic of the Congo in 2012 and 2013 (Fig. 4) (33). The limited sequencevariation (maximum, 1.1%) suggested continued circulation of these viruses during thistime period.

DISCUSSION

The data presented in this study demonstrate the utility of FTA cards as analternative means for the transportation of virologic samples when reverse cold chaintransportation is not available; however, the sensitivity for case confirmation by RT-qPCR and for genotyping was reduced. The lower copy numbers of MeV RNA extractedfrom FTA cards could only partially be explained by the smaller sample volume that wasextracted. Storage of RNA on FTA cards or RNA extraction from FTA cards may affectRNA integrity, which would explain the reduced success rate of genotyping of RNAextracted from FTA cards despite copy numbers above the LOD. Lower copy numbersof the extracted RNA would lead to a higher proportion of false-negative results whenused for case confirmation. However, case confirmation in limited-resource settings

TABLE 4 Comparison of OF and FOF for measles case confirmation by RT-qPCR

FOF PCR result

OF PCR result (no. [%])

Positive Negative

Positive 183 (76.9) 7 (2.9)Negative 31 (13.0) 17 (7.1)Agreement (% [90% CI]) 85.5 (80.2, 89.6) 70.8 (50.8, 85.1)

TABLE 5 Success of MeV genotyping depends on sample type and viral load

Sample

No. genotyping positive results/no. of genotyping attempts (%) for:

Samples with >104 copiesa Samples with <104 copies

TS 22/22 (100) 3/5 (60)OF 19/19 (100) 1/5 (20)FTS 11/13 (86) 1/8 (13)FOF 12/15 (80) 2/7 (29)aCopies of MeV N RNA per RT-PCR.

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such as the Democratic Republic of the Congo is achieved through IgM detection. Theadvantage of using FTA cards for sample transportation would be to expand genotyp-ing. Our data show that FTA cards are suitable for genotyping of outbreaks, whenmultiple samples can be collected, but suggest that this method will have limited utility

FIG 2 Phylogeny based on sequences of MeVs circulating in the Democratic Republic of the Congo in 2014. All sequences were generated in this study except theWHO reference strains (red) and genotype B3 named lineages (blue). Groups corresponding to clusters 1 to 3 and bootstrap values greater than 70% are indicated.Geographic locations are based on province names as used in the Democratic Republic of the Congo in 2014. The tree was constructed using the MEGA 7 neighborjoining algorithm with 1,000 bootstrap replicates. Evolutionary distances were estimated using the p-distance method and are in the units of the number of basedifferences per site.

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for molecular surveillance of sporadic cases of measles in countries that are approach-ing elimination. Currently, only the three regional reference laboratories in WHO/AFRare accredited by WHO to perform molecular methods for measles genotyping (2),which makes it necessary to transport samples over long distances. FTA cards mayimprove virologic surveillance in countries such as the Democratic Republic of theCongo, which are experiencing large outbreaks or endemic circulation of measles andlack the infrastructure to transport samples by reverse cold chain.

All IgM-positive or -indeterminate cases had detectable RNA in at least one sample,and 45 additional IgM-negative cases were confirmed based on RNA detection. Evenwhen only the standard TS was considered, the percentage of suspected cases withpositive RT-qPCR results exceeded that of IgM-positive cases at every time point exceptday seven after rash onset (data not shown). Previous studies have shown that up to23% of confirmed cases are IgM negative within 72 h after rash onset (40). In our study,75% of the IgM-negative cases with serum collected between days 0 and 3 aftersymptom onset had detectable RNA in at least one sample, demonstrating the use ofmolecular methods for case confirmation, especially when samples are collected soonafter rash onset.

The median RNA copy numbers in OF and TS samples were similar, and the positiveagreement of OF with TS was high. Both are excellent choices for virologic samplecollection from suspected measles cases, confirming previously published data (23).The WHO measles and rubella laboratory manual recommends the collection of TSwithin 14 days after symptom onset, but preferably within 7 days, and the collection ofOF within 21 days (41), as decreasing viral loads reduce the likelihood of case confir-mation for later collection dates. The small number of samples that were collectedmore than 7 days after rash onset makes it difficult to draw conclusions, but there wasno indication that OF samples provided an improved likelihood of case confirmation forlate samples.

Transfer of rubella TS or OF to FTA cards resulted in considerable loss of detectableviral RNA, which could lead to misclassification of cases and a reduced likelihood ofobtaining genotypes of circulating viruses. The accumulation of rubella cases in theprovinces of Kasai Occidental (Dibaya) and Sud Kivu (Kabare) indicates that there wererubella outbreaks during the sample collection period. As there is no rubella vaccina-tion program in the Democratic Republic of the Congo (42, 43), rubella remainsendemic (44). Sequence information suggested that the 2BL2c lineage of RuV hascirculated in the Democratic Republic of the Congo since at least 2012 (32).

The use of FTA cards requires training for medical and laboratory personnel. In spiteof supervision by INRB representatives and poster illustrations at each collection site,there were indications of breakdowns in procedures, such as insufficient drying time or

FIG 3 Comparison of RuV RNA loads between sample types. RuV RNA was detected by RT-qPCR assay.Log-transformed copy numbers from 13 patients that were positive by at least one sample type areplotted using Microsoft Excel. Numbers in parentheses indicate numbers of samples that had detectableRuV RNA. Each box represents the interquartile range of the RuV RNA copy numbers recovered from thespecific sample type; the horizontal line in the box indicates the median copy number. The maximal andminimal copy numbers are indicated by the whiskers above and below the box, respectively.

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human errors. Visible mold growth on some FTA cards (16.4% of FTS and 21.8% of FOFcards) indicated that FTA cards were not sufficiently dried. Nevertheless, the mold didnot interfere with the detection of viral RNA (data not shown). Additionally, 5.3% of allsamples showed qualitatively different results than those of other samples from thesame patient, e.g., the FOF was positive with a viral load of at least 1 � 104 copies butthe OF was negative (data not shown). These results indicated that some samples mayhave been collected or labeled incorrectly. On the other hand, only a small fraction ofsamples was excluded because of insufficient RNA quality or quantity; this indicatesthat the collection and transfer of sufficient amounts of viral material was usuallysuccessful.

Sequences that were identical or closely related to the sequences of the named strain,MVi/Harare.ZWE/38.09, were found in all provinces where samples were collected through-out the study period, indicating that this strain was endemic in the Democratic Republic ofthe Congo in 2014. Genotype B3 has been the predominant genotype in sub-Saharan Africa

FIG 4 Phylogeny based on sequences of RuVs circulating in the Democratic Republic of the Congo in2014 (●) and in 2012 to 2013 (�). Remaining sequences are genotype 2B sequences representing the fivegenotype 2B lineages (L0 to L4). Bootstrap values �70% are indicated. The tree was constructed usingthe MEGA6 neighbor joining algorithm with 1,000 bootstrap replicates. Evolutionary distances wereestimated using the p-distance method and are in the units of the number of base differences per site.

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since the outbreaks in South Africa in 2009 (45, 46). Genotype B3 sequences closely relatedto MVi/Harare.ZWE/38.09 have been identified in many countries worldwide (47–50),following the 2013 to 2014 outbreak in the Philippines (51). Two additional clusters ofgenotype B3 appeared to be cocirculating with MVi/Harare.ZWE/38.09 but with morelimited geographic distribution. These results increase our understanding of the variabilityand distribution of genotype B3 in Africa.

Our results suggest that FTA cards may have limited utility for the molecularsurveillance of sporadic cases in countries approaching measles elimination. However,in outbreak situations in resource-limited settings, FTA cards are an attractive alterna-tive transport method for virologic samples when the reverse cold chain is notavailable, facilitating the collection of virologic samples from outbreaks and improvingthe characterization of circulating genotypes.

SUPPLEMENTAL MATERIALSupplemental material for this article may be found at https://doi.org/10.1128/JCM

.00048-19.SUPPLEMENTAL FILE 1, PDF file, 1 MB.

ACKNOWLEDGMENTSWe thank the staff of INRB, especially Yvonne Lay Mowele, Naomie Mitongo

Mwamba, Gloria Ikoli Epanolaka, Seraphine Wanzambi, and Jean Claude MukangalaChanga-Changa, for their technical assistance, Yvonne Villamarzo for technical support,and Howard Gary for statistical analyses. We also thank the WHO regional office and theWHO country office in the Democratic Republic of the Congo for their support.

The findings and conclusions in this report are those of the authors and do notnecessarily represent the official position of the U.S. Centers for Disease Control andPrevention.

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