Accepted Manuscript

Challenges of measles and rubella laboratory diagnostic in the era of elimination

Judith M. Hübschen, Sonja M. Bork, Kevin E. Brown, Annette Mankertz, Sabine

Santibanez, Myriam Ben Mamou, Mick N. Mulders, Claude P. Muller

PII: S1198-743X(17)30214-8
DOI: 10.1016/j.cmi.2017.04.009
Reference: CMI 921

To appear in: Clinical Microbiology and Infection

Received Date: 14 December 2016

Revised Date: 3 April 2017
Accepted Date: 5 April 2017

Please cite this article as: Hübschen JM, Bork SM, Brown KE, Mankertz A, Santibanez S, Ben Mamou
M, Mulders MN, Muller CP, Challenges of measles and rubella laboratory diagnostic in the era of
elimination, Clinical Microbiology and Infection (2017), doi: 10.1016/j.cmi.2017.04.009.

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1 Intended category: Themed Review

3 Challenges of measles and rubella laboratory diagnostic in the era of elimination

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5 Judith M. Hübschen1#, Sonja M. Bork1#, Kevin E. Brown2, Annette Mankertz3, Sabine Santibanez3,

6 Myriam Ben Mamou4, Mick N. Mulders5, Claude P. Muller1, 6 *

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1

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8 WHO European Regional Reference Laboratory for Measles and Rubella, Department of Infection

9 and Immunity, Luxembourg Institute of Health, Esch-Sur-Alzette, Grand Duchy of Luxembourg

2

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10 WHO Global Specialized Laboratory for Measles and Rubella, Virus Reference Department, Public

Health England, London, United Kingdom

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12 WHO European Regional Reference Laboratory for Measles and Rubella, Robert Koch Institute,

Berlin, Germany
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14 Vaccine-preventable Diseases and Immunization, WHO Regional Office for Europe, Copenhagen,
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15 Denmark
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16 Expanded Programme on Immunization, Department of Immunization, Vaccines, and Biologicals,

17 WHO, Geneva, Switzerland

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18 Laboratoire National de Santé, Dudelange, Grand Duchy of Luxembourg
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19 Both authors contributed equally to the manuscript and are considered first authors
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20
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21 * Corresponding author: Prof. Dr. Claude P. Muller, WHO European Regional Reference Laboratory

22 for Measles and Rubella, Infectious Diseases Research Unit, Department of Infection and Immunity,

23 Luxembourg Institute of Health / Laboratoire National de Santé, 29, rue Henri Koch, L-4534 Esch-sur-

24 Alzette, Grand Duchy of Luxembourg, Tel: +352 26970-621 (secretary -620), Fax: +352 26970-660,

25 Email: claude.muller@lih.lu

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27 Running title: Measles and rubella diagnosis in the era of elimination

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29 Abstract

30 Background. The Member States of the WHO European Region adopted the goal of

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31 measles and rubella elimination more than 10 years ago, but so far only 21 of 53 countries

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32 have reached this target. Laboratory investigation of suspected cases is essential to support

33 disease elimination efforts. Therefore WHO maintains a network of accredited laboratories

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34 providing high quality testing. Aims. The present article reviews current measles and rubella

35 laboratory diagnostic methods and describes specific challenges in elimination settings.

36
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Sources. Previously published literature was used to prepare this review. Content.
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37 Laboratory investigation heavily relies on specific IgM serology and increasingly on virus

38 detection by reverse transcription (RT)-PCR, but other methods such as IgG avidity testing
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39 and genetic characterization of virus strains also gain in importance. In elimination settings,
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40 often few samples from suspected cases are available for testing, but testing proficiency
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41 needs to be maintained. The predictive value of an IgM positive result decreases and other

42 rash-fever disease aetiologies become more important. In addition, cases with a rash after
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43 measles/rubella vaccination or with mild disease after waning of vaccine-induced antibodies

44 are seen more often. Thus, it is necessary to perform comprehensive and potentially time-
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45 consuming and costly investigations of every suspected case using quality-controlled

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46 laboratory methods. At the same time rapid feedback to public health officers is required for

47 timely interventions. The introduction of new laboratory methods for comprehensive case

48 investigations may require training of staff under the supervision of WHO accredited reference

49 laboratories and the definition of appropriate test algorithms. Implications. The review

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50 summarizes specific problems and challenges related to measles and rubella laboratory

51 diagnosis in elimination settings and emphasizes that clinical, laboratory and epidemiological

52 data are essential for final case classification and investigation of chains of transmission.

53

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54 Keywords: serology, RT-PCR, diagnosis, virus isolation, ELISA, congenital rubella

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55 syndrome, virus culture, avidity, chain of transmission

56

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57 Introduction

58 More than 10 years ago, the Member States of the WHO European Region (EURO) adopted

59
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the goal of measles and rubella elimination. Based on evidence from 2013 to 2015, only 21 of
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60 53 countries have so far reached this target [1, 2]. Besides a high vaccination coverage
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61 achieved through routine and supplementary immunization activities and high-quality

62 information about immunization benefits and risks, a sensitive surveillance system with
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63 laboratory confirmation of suspected cases is considered crucial to achieve and verify

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64 Regional elimination [1]. The European Measles and Rubella Laboratory Network (LabNet)

65 currently comprises 72 laboratories that ensure high quality laboratory investigation of

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66 suspected cases [3]. All of these laboratories undergo annual WHO accreditation based on a

67 set of performance indicators, including quality assurance and timeliness and completeness
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68 of reporting [3]. While all LabNet laboratories perform measles and/or rubella IgM antibody
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69 detection, an increasing number have introduced virus detection by reverse transcription

70 (RT)-PCR in parallel with serology. In addition, some laboratories perform IgG avidity testing,

71 virus isolation and sequence analysis.

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72 In countries having achieved or approaching measles/rubella elimination, the positive

73 predictive value of IgM serology decreases as more false positives are obtained. Also the

74 number of cases with rash after measles/rubella vaccination or with mild disease after waning

75 of vaccine-induced antibodies will increase [4]. In an elimination setting thorough case

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76 investigations including high quality laboratory testing are crucial. Furthermore, it is important

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77 to use all available information including clinical, epidemiological and patient data, in

78 particular the measles/rubella vaccination status, for case evaluation and classification (Fig.

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79 1). The present article describes current laboratory methods to diagnose measles and rubella

80 infection and highlights diagnostic challenges in pre-elimination and elimination settings as

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encountered in an increasing number of Member States in the WHO European Region and
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82 beyond.
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84 Measles
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85
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86 Clinical and differential diagnosis

87 Measles causes in almost every infected individual symptoms like fever and a maculopapular
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88 rash typically with cough, coryza, or conjunctivitis [1]. Since each of these symptoms is rather

89 nonspecific, clinical diagnosis of measles may be inaccurate. Maculopapular skin rash, for
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90 instance, may be caused by numerous viruses (e.g. rubella virus, parvovirus B19, human
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91 herpesviruses 6 and 7, entero- and adenoviruses, Epstein-Barr virus, cytomegalovirus,

92 coxsackievirus, as well as, in travellers returning from endemic areas, dengue virus,

93 Chikungunya-, Zika-, West Nile-, Ross River-, and Sindbis virus) and the bacterium

94 Streptococcus pyogenes, but also by allergies and drugs [5-7]. In some cases these rashes

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95 may have typical ways of appearing, spreading and fading and a certain distribution, which

96 supports clinical diagnosis [8]. Nevertheless, laboratory confirmation of measles is essential,

97 especially in measles elimination settings [9], when the majority of clinically suspected cases

98 are caused by other aetiologies [10, 11].

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99

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100 Serology

101 The detection of specific anti-measles virus IgM antibodies by ELISA in serum has been the

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102 gold standard for the confirmation of measles. Specimens other than serum like dried blood

103 spots (DBS) or oral fluid, are being used and offer easier alternatives for collection and

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transportation. ELISA tests are fast, inexpensive, reliable, and can be done in a high-
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105 throughput format [12]. Most commonly, indirect assays detect specific antibodies with an

106 immobilized antigen. In capture assays, immobilized anti-human IgM binds IgM antibodies in
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107 the patient specimen. Measles-specific IgM antibodies subsequently bind measles antigen
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108 which is detected by labelled antigen-specific antibodies [13].

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109 Anti-measles virus IgM will persist for at least four weeks after rash [14]. A detectable IgM titer

110 may not have developed until 3 days after onset of rash which may lead to false negative
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111 results [15]. Furthermore, false positive IgM results can occur due to nonspecific reaction,

112 interference of rheumatoid factor, or other underlying conditions such as human parvovirus,
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113 rubella, or human herpesvirus 6 infections [reviewed in 6]. In an elimination setting, where the
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114 positive predictive value of the ELISA assays is lower than in endemic and outbreak

115 situations, false positive results become an important issue necessitating further laboratory

116 testing. To identify false positive results, it may be advisable to retest with another IgM ELISA

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117 kit with a higher specificity and/or a capture assay which may be less prone to interference of

118 rheumatoid factor than indirect assays. False positive results may nevertheless persist [13].

119 Another challenge in an elimination setting is the expected increase in measles infections in

120 previously vaccinated individuals due to the waning of specific antibodies [16]. Such cases

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121 are difficult to diagnose by IgM serology using indirect ELISA assays since IgM titers may be

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122 low or absent [17] and high levels of IgG may interfere with IgM detection [18].

123 In order to exclude potentially false positive or negative IgM results, further laboratory

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124 investigation with either antibody or virus detection methods is required. A significant rise in

125 specific IgG antibody titers in paired acute (collected within 10 days of rash onset) and

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convalescent phase sera (collected 10-30 days after the first sample) can be used to confirm
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127 acute measles infection [14]. While IgG antibody titers are most conveniently determined by
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128 enzyme immunoassays, plaque reduction neutralization assays offer a reliable but

129 cumbersome alternative [4]. Avidity testing may be useful to distinguish between a recent
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130 primary infection characterized by low avidity IgG antibodies and previous contact with either
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131 vaccine or wild-type measles virus [19].

132
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133 Detection of viral RNA

134 Besides IgM serology, detection of viral RNA in clinical specimens by RT-PCR is currently the
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135 most widely used laboratory technique to confirm acute measles virus infection [20]. Suitable
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136 specimens include throat/nose swabs, nasopharyngeal aspirates, oral fluid, urine or

137 peripheral blood mononuclear cells (PBMCs). If a cold chain cannot be maintained during

138 transportation and storage, DBS may be a suitable alternative [21, 22]. Clinical specimens

139 spotted on specific filter paper cards (e.g. FTA™ Elute Micro Cards), which preserve viral

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140 RNA but inactivate the virus, may -like DBS- be shipped at room temperature and as non-

141 infectious material. Measles virus RNA may be detected in serum for up to 7 days after onset

142 of rash and in most other specimens for up to two weeks or even longer [23]. However, to

143 ensure reliable results, samples for virus detection should be collected as early as possible

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144 after rash onset [24].

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145 Conventional end-point RT-PCR is commonly used for generating sequencing templates,

146 while real-time assays are preferred for laboratory diagnosis. Probe based real-time assays

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147 may provide results within 1-2 hours [25], facilitating a timely initiation of control measures if

148 necessary [15]. They require little manipulation, thus reducing the risk of contamination, and

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often target small fragments, thereby allowing detection even in case of partial RNA
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150 degradation. Since in elimination settings most suspected cases are caused by pathogens
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151 other than measles virus, multiplex RT-PCR formats may be advantageous [21, 26]. A critical

152 issue when using end-point or real-time RT-PCR for diagnosis is the quality and the timing of
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153 collection of the clinical specimen. If it has been transported under suboptimal conditions, or
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154 collected late after onset of rash, or the patient has a low level of viremia, a negative RT-PCR

155 result may actually be diagnostically incorrect. Therefore, WHO recommends using IgM
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156 serology as gold standard and using RT-PCR only in combination with serology. However,

157 during the first few days after rash onset, RT-PCR may be diagnostically more useful than
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158 serology [27]. A positive RT-PCR result is diagnostic, a negative outcome needs to be
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159 interpreted with caution.

160 After viral RNA detection, sequencing of the 450 nucleotides of the C-terminal part of the

161 measles virus nucleoprotein gene may be attempted to determine the genotype [28].

162 Information about the genotype and virus variant (e.g. “named strain”) is becoming

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163 increasingly important in elimination settings to help to discriminate between endemic and

164 imported virus variants [28, 29] as well as wild-type and vaccine strains in recently vaccinated

165 persons and to track transmission routes.

166

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167 Potential other diagnostic methods

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168 Measles virus may be isolated from samples such as throat/nasal swabs, nasopharyngeal

169 aspirates, urine or PBMCs in cell cultures of B95a (a marmoset lymphoblastoid cell line) or

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170 Vero/hSLAM cells (African Green Monkey kidney cells transfected with the human CDw150

171 (SLAM) measles virus receptor) [30], for instance. Since the latter are, in contrast to B95a

172
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cells, not persistently infected with Epstein-Barr virus and are thus not considered infectious
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173 material per se, show a similar sensitivity as B95a and also allow isolation of rubella virus,

174 they are recommended by WHO for use within the Global Measles Rubella Laboratory
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175 Network [14]. After infection of cell cultures with measles virus it may take several days until a
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176 cytopathic effect becomes visible [31]. The method is also less sensitive than RT-PCR and is
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177 no longer routinely used for diagnosis. It is mostly applied when high viral titers or sufficient

178 material for in-depth virus characterization are required.

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179 Other recently described methods for diagnosis of measles, such as automatable focus

180 reduction neutralization tests (AFRNT) [32], chemiluminescent immunosorbant assays (CLIA)
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181 [33], or loop-mediated isothermal amplification (LAMP) [34], are used by some laboratories.
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182

183 Rubella

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185 Clinical and differential diagnosis

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186 Rubella is characterized by maculopapular rash and cervical, suboccipital or postauricular

187 adenopathy or arthralgia/arthritis [1]. With up to 50% of infections being asymptomatic or

188 subclinical and with symptoms similar to those caused by measles virus and other rash

189 aetiologies, clinical diagnosis of rubella is unreliable [35]. Parvovirus B19 infection is

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190 especially difficult to distinguish from rubella, since fever, rash and joint symptoms are

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191 common in both infections [36]. Therefore, suspected cases should be confirmed by

192 laboratory testing. This is particularly critical during pregnancy to evaluate the risk of

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193 congenital rubella syndrome (CRS) [37]. Details about clinical and laboratory diagnosis of

194 CRS may be found elsewhere [e.g. 36].

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196 Serology

197 Detection of rubella-specific IgM in serum is the standard method for laboratory diagnosis of
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198 rubella [38]. Alternatively, oral fluid [39] or DBS [20] can be used. Rubella-specific IgM
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199 antibodies may not be detectable until 4 days after rash onset [40] and in rare cases patients
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200 may even fail to produce detectable IgM antibodies [41]. Normally, IgM antibodies persist for

201 at least 8 weeks and occasionally much longer, particularly after vaccination, where IgM
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202 antibodies may persist for more than 6 months [42]. Most commercial assays use an indirect

203 format, with immobilized whole rubella virus and detection of specific IgM by an anti-human
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204 IgM conjugate. However, these assays may give false positive IgM-results due to cross-
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205 reactive IgM antibodies from other infections (i.e. enterovirus and adenovirus infections),

206 interference of rheumatoid factor (i.e. as part of the immune response to parvovirus B19

207 infection or in patients with other causes of arthropathy) or cross-reacting antibodies to the

208 membrane component of the whole virus preparation in sera from pregnant women [11, 43].

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209 IgM capture assays are more specific, but false positive reactions are still possible [12].

210 Especially during pregnancy it is essential to distinguish primary infections from persisting IgM

211 antibodies and rare cases of re-infection. Due to the problem of cross-reacting antibodies to

212 the membrane component of the whole virus preparation in sera from pregnant women,

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213 assays using recombinant rubella antigens such as C, E1 and E2 proteins may be more

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214 specific during pregnancy. Since antibodies against the rubella E2 protein are not detectable

215 until 3-4 months post-infection, they exclude acute primary infection [43].

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216 As the number of rubella cases decreases, the predictive value of an IgM positive result also

217 decreases, and thus confirmation of IgM positive samples becomes important [44].

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Seroconversion or a significant increase in rubella IgG titer in paired acute and convalescent
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219 phase sera tested in the same assay are indicative of an acute infection [14].
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220 Recent primary rubella infections can be confirmed by rubella-specific low avidity IgG.

221 Commercial rubella avidity assays are based on specific IgG binding in the presence or
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222 absence of a mild chaotropic agent [45]. Low avidity antibodies normally persist for 2-3
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223 months after a primary infection and sometimes even much longer after first vaccination [35].

224
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225 Detection of viral RNA

226 As for measles, real-time methods including TaqMan assays are the preferred RT-PCRs for
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227 laboratory confirmation of rubella infection. Conventional endpoint assays, in particular nested
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228 formats, are mostly used for amplifying products for sequencing. While these nested RT-

229 PCRs are very sensitive and allow amplification also in case of low viral load and from

230 suboptimal specimens such as serum, they require strict contamination control measures

231 [46]. The same clinical specimens may be used for measles and rubella virus detection,

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232 facilitating simultaneous or subsequent investigation. Early and appropriate sample collection

233 as well as adequate transportation and storage are essential to avoid false negative results.

234 Rubella virus viremia is low and frequently no other specimen besides serum is available for

235 investigation. The diagnostic RT-PCR assay should therefore be very sensitive to reliably

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236 detect rubella virus [47], particularly when the patient is pregnant and at risk for congenital

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237 infection. As with measles, rubella genotype data, based on 739 nucleotides of the viral E1

238 gene [48], is critical to understand the epidemiology of the virus and to verify elimination [49].

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239

240 Potential other diagnostic methods

241
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Rubella virus grows in different cell lines such as rabbit kidney RK13, Statens Seruminstitut
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242 Rabbit Cornea SIRC, baby hamster kidney cells, Vero and Vero/hSLAM cells. In contrast to

243 measles virus, wild-type rubella virus seldom produces a distinct cytopathic effect [50]. Even
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244 laboratory-adapted strains usually take 5 days at 35°C to develop plaques in a conventional
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245 plaque assay [51]. Thus, additional tests such as RT-PCR, immunofluorescence or
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246 immunocolorimetric assays may be required to confirm virus isolation. With the latter assay,

247 for instance, results can be obtained within 3 days, when stained small foci of infection
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248 become visible [31]. This method is as sensitive as immunofluorescence assays for

249 confirmation of virus isolation and may also be used in neutralization assays to detect
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250 neutralizing antibodies [51]. Since rubella virus is often present in low quantities in clinical
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251 samples and the genetic region recommended for genotyping is larger than for measles virus

252 [14], isolation in cell culture may be required to obtain sequence information for virus

253 characterization.

254

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255 Discussion

256 In an elimination setting, where every single case of measles or rubella needs to be identified,

257 sensitive, specific, high quality and standardized tests are required to support swift public

258 health interventions. It is the role of the local surveillance systems to guarantee that adequate

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259 clinical specimens are collected in the correct way and at the correct time point in the course

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260 of infection. Especially for specimens intended for RT-PCR or virus isolation, proper specimen

261 transportation and storage are critical to prevent specimen degradation [52].

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262 While appropriate controls are essential for all diagnostic steps, RT-PCR-based methods

263 benefit from including an internal positive control such as RNase P to assess sample quality

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and the presence of inhibitors in the reaction [53]. Inhibition may also be detected by spiked-
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265 in controls. Both for serology and molecular assays, standardized positive controls allow
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266 detection of inter-assay variability and deviations over time due to changes in reagent lots or

267 staff. Since clinical samples from suspected measles/rubella cases may be rare in elimination
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268 settings, maintaining testing proficiency may rely on regular test runs on stored material or
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269 practice panels. For the establishment of additional diagnostic tools, training may be required

270 and clear testing algorithms must be defined. The importance of these quality-related aspects
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271 may require the supervision of laboratories performing measles and/or rubella diagnosis by

272 WHO accredited reference laboratories [14]. A performance indicator to monitor the
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273 performance of surveillance and laboratory has been introduced by WHO in the Framework
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274 for Elimination [54]. To ascertain a sufficiently sensitive surveillance system, a country has to

275 investigate and reject at least 2 non measles non rubella cases per 100,000 population. This

276 indicator is not always easy to determine if many private and hospital laboratories are

277 involved in measles and rubella diagnosis.

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278 In elimination settings, rapid feedback to the public health officers is crucial for timely

279 interventions [15], but at the same time the investigation of sporadic cases and chains of

280 transmission will become increasingly complex, time-consuming and costly. Some

281 laboratories may even consider establishing diagnostic tests for rash-fever disease

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282 aetiologies that are becoming more important with a declining incidence of measles and

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283 rubella. The identification of other pathogens supports the rejection of suspected cases as

284 non-measles and non-rubella [11].

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285 Virus characterization by sequencing and timely sharing of the data via the WHO-supported

286 databases MeaNS (www.who-measles.org) and RubeNS (www.who-rubella.org) has

287
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demonstrated its importance in analysing a country’s achievement in verifying elimination and
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288 to differentiate between endemic and imported as well as wild-type and vaccine-derived
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289 strains [55]. To obtain genotyping information supporting interruption of endemic virus

290 transmission is considered to be an essential criterion for verification of measles/rubella

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291 elimination in EURO [1]. In general, laboratory results should always be interpreted together
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292 with all other clinical and epidemiological information for adequate case classification and

293 characterization of chains of transmission.

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294

295 Transparency declaration

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296 The authors declare no conflicts of interest. No external funding was received to write this
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297 review.

298

299 References

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447

448 Legend to Figure

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449 Fig. 1. Examples of information potentially influencing measles/rubella case evaluation and

450 classification

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Laboratory Information
e.g.:
• Appropriate specimen
• Collection timepoint
• Sample quality

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• Serological test results (IgM, IgG, IgG-avidity)
• Virus detection by PCR or virus isolation

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• Sequence and genotype information
• Detection of other rash-fever pathogens

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Clinical Information Epidemiological Information

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e.g.: Case Evaluation and e.g.:

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• Date of onset of prodromal symptoms Classification * • Travel history prior to onset of symptoms
• Date of onset of rash e.g.: • Contact with recent travelers from endemic
• Appearance and distribution of rash countries

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• Endemic, imported, import-
• Clinical history related • Contact with rash-fever patients
• Disease complications • Clinically compatible, • Link to outbreaks/laboratory confirmed cases

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epidemiologically linked, • Status of measles/rubella elimination in the

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EP laboratory-confirmed country/region

Patient Information
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e.g.:
• Gender and age in relation to local
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immunization schedules
• Place of residence
• Vaccination history and status

er information on WHO case classifications, please refer to:

euro.who.int/__data/assets/pdf_file/0009/247356/Eliminating-measles-and-rubella-Framework-for-the-verification-process-in-the-WHO-European-Region.pdf?ua=1