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FIGURE 1. Pattern of test results among patients with wild measles virus infectior able to the population (6), thereby

by day from rash onset and type of sampling method used — WHO Measles and

Rubella Laboratory Network* enabling health-care workers to obtain more complete sampling for suspected cases.

/ Evaluations Comparing

IgGt: Serum/DBS OF

IgM". Serum DBS OF Alternative Diagnostic

.. Virus detection"": OF Samples with Serum

Virus detection": DES

Virus culture Based Diagnostics

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Since 2001, LabNet reference labo-
ratories in Australia, Cote d'Ivoire,
Netherlands, Turkey, Uganda, the
UK, and the United States have been

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60 working to 1) determine IgM and

Days from rash onsetos RNA stability in DBS and OF

* Illustrative schematic based on data presented at the Measles and Rubella Alternative Sampling samples and 2) optimize the meth- Techniques Review Meeting, convened in Geneva, Switzerland, in June 2007. ods for IgM antibody assay and pro

Immunoglobulin G.

§ Dried blood spots. tocols for RNA detection in DBS and 1 Oral fluid. OF samples (8-10). This work has ** Immunoglobulin M.

ft Virus RNA detection by conventional, nested, or real-time reverse transcription-polymerase provided data on sensitivity and speci

chain reaction. ficity of OF and DBS samples com

$8 Incubation period: approximately 14 days. pared with serum and also has identified logistic challenges in implementing alternative

RNA in serum and DBS was shown to be possible with sampling techniques. Three different types of data were

nested or real-time RT-PCR (but not conventional RT-PCR available for review during the ad hoc meeting. First,

if samples are collected within 5-7 days after rash onset. beginning in 2001, LabNet laboratories conducted stud

This procedure has proven invaluable for collecting viral ies that collected OF, DBS, and corresponding serum

sequence information where urine or throat swabs were not samples from persons with suspected measles or rubella

available. In the MMR surveillance program in the l'K. during outbreaks and tested the samples for the presence

using OF, the rate of measles RNA detection by nested of measles- or rubella-specific IgM antibodies. Second,

RT-PCR ranged from 80% to 90% when collected during LabNet reviewed data from the MMR surveillance program

the first week after rash onset, and reached 50% at 3-4

weeks after rash onset. Conventional RT-PCR was sensitive in the UK, where 1,000–3,000 OF samples have been collected annually during the past decade. Third, LabNet re

for up to 2 weeks after rash onset, but was still considered viewed data from seven countries in the WHO African

useful. For rubella, testing for both IgM and RNA in OF Region that used DBS sampling methods for routine

samples substantially increased the sensitivity of surveilmeasles and rubella surveillance during 2005–2007. DBS

lance for confirming cases during the first 4–5 days after was either the only sample collected (Sierra Leone) or was

rash onset, when many rubella cases are not yet IgM posicollected in conjunction with routine serum collection

tive. Results of evaluations comparing OF and DBS with (Burkina Faso, the Democratic Republic of Congo, Ethio

serum sampling indicated that OF and DBS sampling have pia, Ghana, Senegal, and Zambia). Standard protocols for

a potential role in improving measles and rubella surveilsample collection and laboratory testing recommended by

lance. Compared with serum collection, these sampling LabNet were used (2).

procedures provide: Data from all three sources indicated that the sensitivity

Equivalent sensitivity and specificity for specific IgM and specificity of DBS and OF for detecting measles and

detection, although moderately reduced sensitivity for rubella virus-specific IgM parallels that of serum; however,

detecting rubella virus-specific IgM in OF samples

. a moderate decline in sensitivity for detecting rubella virus,

• Simplified sample collection, although training is specific IgM in OF during the first 4-5 days after disease

required. onset was observed (Figures 1 and 2; Table). Detection of

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FIGURE 2. Pattern of test results among patients with wild rubella virus infection, by because DBS avoids venipuncture

day from rash onset and type of sampling method used WHO Measles and Rubella

Laboratory Network and OF is noninvasive. Stability without refrigeration for periods of up to 7 days (OF) or longer (DBS).

7Equivalent cost for collection,

extraction, and testing. ... Potential to substantially reduce

IgG+: Serum/DBS transport costs through avoiding

IgM?: Serum/DBS refrigeration.

IgM: OF Ability to detect both specific IgM and RNA in the same sample. Of can extend the opportunity for RNA detection

3 after rash onset.

Days from rash onsetos Equivalent sensitivity and speci- Illustrative schematic based on data presented at the Measles and Rubella Alternative Sampling .*. ficity for IgG detection and con

Techniques Review Meeting, convened in Geneva, Switzerland, in June 2007.

+ Immunoglobulin G. sequent versatility for use in

§ Dried blood spots. seroepidemiology studies.

1 Immunoglobulin M.

Oral fluid. However, use of OF and DBS sam

it Virus RNA detection by conventional, nested, or real-time reverse transcription-polymerase ing also has some disadvantages chain reaction.

$8 Incubation period: 14–17 days. mpared with serum collection, in rticular: · Collection devices are not commonly available and • Extraction procedures for DBS and OF require more

would need to be provided to health-care facilities by time of technicians. the surveillance program.

• External quality-assurance programs, such as those cur• Volume of DBS might be inadequate unless staff are rently required for testing of serum, have yet to be fully trained in sample collection.

established for OF and DBS.

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BLE. Percentage of patients testing positive for wild measles and rubella virus infection, by time of specimen collection, type of ecimen, and type of sampling method used — WHO Measles and Rubella Laboratory Network* Time of collection

Serum (%) Dried blood spots (%) Oral fluid (%) {asles Early (day 0-3)

60-70
60–70

60-70
Intermediate (day 4-14)

90-100
90–100

90-100
Late (day 15–28)

100
100

100 --irus detection (RT-PCR$) Early (day 0–3)

<10
<25

>80
Intermediate (day 4-14)

<1 Late (day 15–28)

0

0

<20
ibella
Early (day 0–3)

50
50

40
Intermediate (day 4-14)

60-90
60-90

50-90
Late (day 15–28)

100
100

100 irus detection (RT-PCR) Early (day 0–3)

-1

20

60-70
Intermediate (day 4-14)

-1
_1

50
Late (day 15–28)

1
-1

_11 ased on data presented at the Meeting on the Use of Alternative Sampling Techniques for Measles and Rubella Surveillance, convened in Geneva, witzerland, in June 2007. nmunoglobulin M. irus RNA detection by conventional, nested, or real-time reverse transcription-polymerase chain reaction. ata are insufficient for meaningful analysis.

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Recommendations

Having considered the evidence described in this report, participants in the ad hoc meeting made the following recommendations.

No single alternative sampling technique has been shown to be optimal for surveillance under every circumstance, and serum should still be considered the “gold standard” for IgM detection. However, DBS and OF sampling techniques are viable options for measles and rubella surveillance (5–10), especially where challenges with specimen transport or refrigeration exist or where patients might resist venipuncture. Alternative sampling techniques would not adversely affect routine measles and rubella surveillance (provided adequate training and resources are provided) and might enhance surveillance through:

• More acceptable noninvasive methods (OF). • Reduced transport costs (DBS and OF). • Enhanced ability to conduct molecular surveillance (OF

and DBS RNA). • Enhanced sensitivity of rubella case confirmation dur

ing the first 4–5 days after rash onset (OF RNA). • Offering a confirmatory option for questionable serum

IgM results during the early stage of disease for both

measles and rubella (OF RNA). Regions in the elimination phase that already have established a serum-based rash illness surveillance system would not likely benefit from changing to DBS or OF sampling methods except in special circumstances, such as in settings where:

Timely specimen transport from remote or difficultto-access areas to the laboratory conducting the sero

logic analysis is especially difficult. • Collection of OF in addition to serum might improve

efficiency of case identification and virologic surveillance by enabling detection of viral RNA from disease

with a combined MMR vaccine, have been critical in achie ing that public health success. At present, routine mease and rubella surveillance in the United States will contin. to rely upon already established diagnostic method including serum-based assays for detection of virus-specif. antibodies and on nasopharyngeal swab or urine sampk for virus detection. References 1. World Health Organization. Global measles and rubella laborato

network-update. Wkly Epidemiol Rec 2005;80:384-8. 2. World Health Organization. Manual for the laboratory diagnosis :

measles and rubella infection, 2nd ed. Geneva, Switzerland: Work Health Organization; 2007. WHO/IVB/07.01. Available at herp

www.who.int/immunization_monitoring/LabManualFinal.pdf. 3. Ibrahim SA, Abdallah A, Saleh EA, Osterhaus ADM, De Swart

Measles virus-specific antibody levels in Sudanese infants: a prospe.tive study using filter paper blood samples. Epidemiol Inter

2006;1 34:79-85. 4. Riddell MA, Byrnes GB, Leydon JA, Kelly HA. Dried venous blood

samples for the detection and quantification of measles IgG using a commercial enzyme immunoassay. Bull World Health Orga

2003;81:10. 5. El Mubarak HS, Yüksel S, Mustafa OM, Ibrahim SA, Osterhaus AD de Swart RL. Surveillance of measles in the Sudan using

filter

paper blood samples. J Med Virol 2004;73:624-30. 6. Vyse AJ, Gay NJ, White JM, et al. Evolution of surveillance of measles

mumps, and rubella in England and Wales: providing the platform :!

evidence-based vaccination policy. Epidemiol Rev 2002;24:125– ší. 7. Vyse AJ, Jin L. An RT-PCR assay using oral fluid samples to detec

rubella virus genome for epidemiological surveillance. Mol Cell Probes

2002;16:93-7. 8. De Swart RL, Nur Y, Abdallah A, et al. Combination of reverse tran

scriptase PCR analysis and immunoglobulin M detection on Inte? paper blood samples allows diagnostic and epidemiological studies ei

measles. J Clin Microbiol 2001;39:270–3. 9. Riddell MA, Leydon JA, Catton MG, et al. Detection of measles virus

specific immunoglobulin M in dried venous blood samples by using a

commercial enzyme immunoassay. J Clin Microbiol 2002:40:5–9. 10. Helfand RF, Cabezas C, Abernathy E, et al. Dried blood spots ver

sera for detection of rubella virus-specific immunoglobulin M DEN and IgG in samples collected during a rubella outbreak in Peru. Ü. Vaccine Immunol 2007;14:1522–5.

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iger-stick whole-blood specimens using the OraQuick®

and test (OraSure Technologies, Bethlehem, Pennsylvania). : March 2005, the clinics replaced finger-stick whole-blood sting with oral fluid testing with the OraQuick Advance pid HIV-1/2 Antibody Test.* The clinics use Western blot nfirmatory tests on serum to confirm all whole-blood or al fluid reactive (i.e., preliminary positive) rapid tests. In e 2005, an unexpected increase in the number of falsesitive oral fluid tests occurred, but the increase subsided er several months. In December 2005, while the cluster false-positive oral fluid test results was being investigated, e NYC DOHMH Bureau of STD Control suspended oral iid testing in the clinics for 3 weeks and replaced it with iger-stick whole-blood rapid testing, which produced no se-positive test results. On December 21, 2005, NYC OHMH resumed oral fluid rapid testing but also introiced the use of immediate follow-up finger-stick wholepod testing, using a second OraQuick test, after any reactive al fluid test result. In late 2007, another larger increase in e incidence of false-positive oral fluid rapid test results was served. The cause for the episodic increases in false-posire oral fluid tests has not yet been determined. NYC OHMH has again suspended the use of oral fluid testing

STD clinics, and finger-stick whole-blood testing is the ily rapid HIV test being used in this setting. These findgs underscore the importance of confirming all reactive IV tests, both from oral fluid and whole-blood specimens.

addition, the results suggest that the NYC DOHMH rategy of following up reactive oral fluid test results with

immediate finger-stick whole-blood test reduced the numr of apparent false-positive oral fluid test results and might

a useful strategy in other settings and locations. The NYC DOHMH Bureau of STD Control routinely fers STD and HIV screening to all patients during the 'proximately 115,000 annual visits to the 10 STD clin

operated by the city. In 2003, 33,375 conventional (i.e., et rapid) HIV tests were performed. A total of 552 (1.6%) ere positive; 79% of all patients tested received their test sults. In 2004, after on-site finger-stick whole-blood rapid , IV testing was initiated with the OraQuick test, HIV sting at the clinics increased 14% to 38,092 tests, and ceipt of results increased to 88% for HIV-positive and 5% for HIV-negative patients. On average, during nuary 2004-February 2005, fewer than one falseositive finger-stick whole-blood rapid test occurred onthly. After oral fluid rapid HIV testing began in March

2005, overall test volume increased an additional 24%, to 47,204 tests in 2005. This upward trend in testing has continued (Figure 1); in 2007, the STD clinics performed 60,281 HIV tests, of which 607 (1.0%) were confirmed positive.

In the first 7 months after oral fluid testing was introduced, 35 (0.16%) of 21,722 tests were false positive by Western blot, consistent with the 99.8% (95% confidence interval [CI] = 99.6%-99.9%) specificity claim by the manufacturer in the product package insert (1). However, in October 2005, staff members at the clinics noticed an increase in the number of false-positive oral fluid test results each month. From an average of five false-positive tests per month, the monthly number of false-positive tests increased to 11 (0.27% of 4,024 tests) in October 2005 and to 36 (0.97% of 3,735 tests) in November 2005 (with a specificity of 99.03%, lower than the lower limit of the manufacturer's CI specifications) (Figure 1). An investigation detected no consistent relation between false-positive results and test-kit handling, storage conditions, or lot numbers or between false-positive results and clinic sites, test operators, or patient characteristics.

Despite the increased number of false-positive results, testing with the noninvasive oral fluid specimens was popular with clinic patients and more convenient for staff members; therefore, the NYC DOHMH continued offering oral fluid rapid HIV testing while attempting to minimize the adverse effects of false-positive test results. In late December 2005, a revised strategy was implemented at the clinics by continuing to offer oral fluid rapid tests but immediately following reactive oral fluid tests with a second OraQuick test on finger-stick whole-blood specimens. Both test results were documented in the medical record. Counselors continued to explain to patients that any reactive rapid tests required Western blot confirmation but also emphasized that discordant oral fluid and whole-blood test results were likely to be false positive. By February 2006, an oral fluid test specificity of 99.65% was observed, within the CI of the manufacturer's specifications.

Another persistent increase in false-positive oral fluid test results began in late 2007. Beginning in November 2007, the number of false-positive oral fluid tests increased from 23 (0.51% of 4,503 tests) to a peak of 54 (1.11% of 4,858 tests) in February 2008 (Figure 1). During November 2007–April 2008, the monthly specificity of the oral fluid test ranged from 98.88%-99.49%. In May 2008, fewer false-positive tests occurred; in that month, five (0.11% of 4,749 oral fluid tests) were found to be false positive (specificity: 99.89%).

he OraQuick rapid HIV test can be used to test either blood (finger-stick or enipuncture whole-blood or plasma specimens) or oral fluid.

FIGURE 1. Total number of oral fluid rapid human immunodeficiency virus (HIV) tests administered and number of actual and expected false-positive results,* by month and year - New York City, March 2005-May 2008 $ 60

7
False-positive tests

Total tests
50
Expected false-positive tests

6
.: 95% confidence interval (for expected false-positive tests)

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* As confirmed by Western blot performed on serum. Expected number of false-positive tests and corresponding 95% confidence intervals calculated

based on number of oral fluid tests performed monthly and manufacturer's claim for specificity with oral fluid (Orasure Technologies, Inc., OraQuick? Advance Rapid HIV-1/2 Antibody Test customer letter and package insert. Available at http://www.orasure.com/uploaded/398.pdf). t Among patients tested in 10 sexually transmitted disease clinics. $ Oral fluid rapid HIV tests were introduced in March 2005. They were suspended for 3 weeks in December 2005 and replaced by finger-stick whole-bloot testing.

During this second instance of increasing numbers of false-positive oral fluid tests, the clinics continued offering immediate follow-up finger-stick whole-blood rapid tests for all patients with reactive oral fluid tests. The usefulness of the NYC DOHMH policy was affirmed by the strong correlation between results from whole-blood rapid tests and confirmatory Western blot tests. During December 2005–May 2008, 1,720 patients had reactive oral fluid rapid tests, and definitive Western blot results were recorded for 1,664 (Figure 2). Missing Western blot results (24 patients) and inconclusive Western blot results (32 patients) were excluded from additional analysis. Of these 1,664 patients, 1,194 also provided a finger-stick specimen; 850 (71.2%) had a reactive finger-stick test, of whom 840 (98.8%) were positive by Western blot. Only one (0.3%) of 344 patients with a reactive oral fluid and negative finger-stick whole-blood rapid test was positive by Western blot.

Despite the NYC DOHMH policy that STD clinics should retest using whole-blood specimens after reactive oral fluid tests, 550 patients with reactive oral fluid results

did not receive a finger-stick test. For 80 of these patient. the test was ordered but not completed; of these, 77 (96.3% had a positive serum Western blot result. A total of 40 (28.2%) patients with reactive oral fluid tests declined the finger-stick test. Of these, 455 (96.8%) were confirmed positive by serum Western blot, compared with 850 (71.2%) of the 1,194 patients who agreed to a finger-stick test. Additional investigation indicated that 29% of patients with a reactive oral fluid test result who then declined the finger-stick test had been reported previously as HIV-positive to the local HIV/AIDS Reporting System compared with 21% of patients who agreed to a follow-up finger-stick test.

* Before patients were examined by a clinician, STD clinic staff members dreu nav

vials of blood from all patients who visited the clinics (one for syphilis testing and on for confirmation of HIV, if needed). Clinic providers offered the HIV test 102 patients; if accepted, providers requested the signed consent form required by the state of New York, and, when the oral fluid test was being used, they conducted the oral fluid rapid HIV test. Patients with reactive oral fluid tests were offered the fingerstick whole-blood test. The clinics were able to obtain confirmation of resuls to patients who refused the finger-stick test because the initially drawn tube of blocks sent routinely for Western blot confirmation of all reactive tests.

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