de Beer, M

Smit, EJ

Steele, AD

English

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THE SOUTHERN AFRICAN JOURNAL Of HIV MEOICINE -------------- MAY 200 IFROM THE LABinaccurate owing to different infectionrates and other co-factors in individualsother than pregnant women attendingantenatal clinics.Additional sero-epidemiological surveysare frequently performed in order toguide programme management, as theyhelp to formulate prevention strategiesand monitor intervention efforts incertain populations. High costs and fearof breaches in confidentiality reduce thestatistical power of these results inmany cases.The method employed for DBS testing using a modifiedSerodia particle agglutination assay [Fujirebio Inc., Japan)involves the following procedure. A drop of blood sampleis collected onto No. 3 Whatman blotting paper and left toThe use of dried blood spots (DBS) forthe testing of HIV-1 antibodies has beendescribed since 1988.' Numerousstudies have proved that this method ofprevalence testing has high sensitivityand specificity and is easy to use, as wellas having the advantage of being lessexpensive than conventional tests onwhole blood.~' The elution from thespots can be tested for HIV RNA, DNA orantibodies to HIV. as they all seem to bestable for long periods of time evenwhen stored at room temperature.'" Amodified particle agglutination anti-HIV-1 testing method'is particularly useful as it reduces the cost of a normalELlSA kit to almost one-tenth so the cost per test isapproximately R2.50.E J Smit, MB ChB, DTM&H, MMed (Cfin Viral), FCPath (SA) (Clin Viral)M de Beer, Not Dip Med Lab TechA D Steele, PhDDepartment of Virology and MRC Diarrhoea Pathogens Research Unit, Medical University of Southern Africa,PO Medunsa, 0204The AIDS epidemic in South Africa hasnow reached the alarming level atwhich almost 1 in every 10 SouthAfricans is infected. This figure isextrapolated from the 10th NationalAntenatal HIV Survey, which wasconducted during October 1999,' andtranslates into an absolute figure ofaround 4.2 million people infectedwith HIV in South Africa, one of thehighest in the world. It is thereforeimportant that educational andawareness programmes reduce therate of new infections. The importanceof finding the right programme for theright targeted population lies in thefact that an estimated1 600 new infections occur daily inSouth Africa. New 'incidence'enzyme-linked immunosorbent assays[ELlSAs) can be a great help inevaluating the success of anyintervention programme,' but theirperformance has not yet beenreported on in South Africa. Thealternative of using figures obtainedfrom the Department of Health'snational HIV seroprevalence surveys of women attendingantenatal clinics is not a satisfactory solution as these aretoo crude for true representation or comparison ofintervention studies. The 1999 national survey used 487sites as representative of the country as a whole, andinformation gained from these clearly showed that theepidemic differs in different geographical regions in SouthAfrica. Calculating the total estimated HIV- positive males,children and elderly from these figures would be grosslyTHE USE OF DRIED BLOOD SPOTSAND A MODIFIED PARTICLEAGGLUTINATION TEST FORCOST-EFFECTIVE SERO-EPIDEMIOLOGICAL HIV SURVEYSTHt SOUTht;{N 4FR.ICAN JOUR-NAl OF HIV Ivl~OICIN[ ---------------IvIAy 200 Idry. DBS samples can be stored in sealed plastic bags atroom temperature, alt:lough if a prolonged period ofstorage is envisaged storage at -20"C is advisable. At thetime of testing a 5 mm punch of each DBS is placed in aflat-bottom microtitre plate. The sample is eluted byadding 100 ~I TPHA buffer (Fujirebio) to each well, and theplate is incubated at 4°C overnight. A 1:5 dilution [5 ~I +20 ~I) of the eluted sample is made in a V-shapedmicrotitre plate in TPHA buffer. Gelatin particles sensitisedwith inactivated HIV- 1 antigens are reconstitutedaccording to the manufacturer's instructions (Fujirebo) and25 ~I of a 1:10 dilution of the reconstituted particles isadded to each well. Apositive and anegative control bloodspot, treated in the same way as the tEst samples, areincluded in each plate. The plate is covered, shaken and leftfor 3 hours on a vibration free surface at roomtemperature. Results are read visually. A positive result isrecorded on observation of a tight discrete agglutinationpattern. Non-reactivity is identified by the presence of asmall definite pellet at the bottom of the well [Fig. 1).Fig. 1. A microtirre plate with positive (fuzzy centre) and negative[tight red bu[mn) results.We have extensively used and tested this modified particleagglutination in various epidemiological studies. Thepatient populations tested included infants in the neonatalunit of Ga-Rankuwa Hospital over a 3-year period. Thedrawing of blood from neonates requires a speciallytrained health care worker [HCW], while a DSS from a heelprick can be obtained easily. The second group of patientstested [unlinked anonymous testing) were those whoattended the antenatal clinic [ANC) and sexuallytransmitted disease clinic [STD) at two different locationsserved by the Centurion Municipal Department of Healthduring the period January - Dctober 1999. The third groupof patients participated in an anonymous unlinkedseroprevalence survey. These were inpatients at Ga-Rankuwa Hospital, a large 1 400-bed referral hospital onthe border of three provinces. In the course of 1 day ateam of volunteer hospital HCWs briefed hospital patientsin groups of ± 15 patients at a time about HIV [ways oftransmission and prevention). The patients were asked toconsent to a finger prick, which was done by a health careworker. Thereafter the patients had to blot their blood onblotting paper, let it dry, and deposit it into a collection box.The high participation percentage of 90.8% [912/1004) inthis 1-day point prevalence study was largely due to theTacts that a small amount of blood was asked for and thatit was obvious to most patients that [he method employedconferred total confidentiality.Dne hundred consecutive HIV antibody requests to ourdiagnostic laboratory were analysed as a quality controlprocedure using DBS and the modified Serodia panicleagglutination assay [Fujirebio). All 38 blood samples thattested positive with our routine diagnostic HIV 1/2/0Axsym assay (Abbott) also tested positive with the modifiedSerodia particle agglutination assay, i.e.100% sensitivity.Of the 62 negative results obtained by the HIV 1/2/0 Axsymassay, 1 was falsely detected as positive by the modifiedSerodia particle agglutination assay, i.e. 98.4% specificity.We therefore recommend that this test be used forunlinked sero-epidemiological surveys only and not fordiagnostic purposes.In conclusion, the use of DBS as an alternative method forsero-epidemiological surveys is not only relatively cheap,but can be performed by minimally trained field workersand is perceived by patients to be more confidential thanother methods if performed in the right way. Samples canbe stored at room temperature and mailed in the standardmail service in a sturdy envelope.DSS methods have proved to be safe, easy to perform andreliable and provide a useful method for large-scale sero-epidemiological surveys especially in developing countriesand isolated rural regions where resources are limited.REFERENCES1. Dep3r:me~t of Health. SlmmafY Rr:oort, National HIV SerolJ'e',c!erce SrJrw:y 0"V'!omen A-::er.ding AnrenorcJ Oinics in South Africa, 1999. Pre:o'"a: DOH, 2000.2. Janssen RS, Sanen GA, Srramer Sl ~'al. Nl"w ,es(ng stml.:'gy :0 cW'cr ear! HIV-1:nrl.:'clior "or USI.:' in ir:cidence estim;les ara "or c ;n,c~ pJf;Joses, JAM/-, 1998; 280:-1-2-~.H~nron WH, leNS OS, Powell MI<. A (jua ity assurancl': :lrcgr~r"" ·or hUrl'ar:mr"'~rodeficiency ~'rus se'opositi~ity screenirg 0" oried-oooo SpOt s~ecml':nsInfw Coruel Hosp Eo'de'1ioI1988: 9(8): 361.-1-, ?re',a enet" o· fl"\a!erral HN ir.feefon o"seo on ur :rkl"d ano~yr"'Ous Itstirg 0"'le,',oorn bab:es (see comrrerrsJ. Lancer 1990; 335: 516-5195. Eoi ot F, Feelers M, Ko,:" A. Deapor:e E. Pre"a e"ce o· nu"",," 'rr'murodeO"cency'irus among pa:'ems witn tuberc osis ir Sellfa Leore, estao:isred fro"l 0' eaolood spo:s on f lel paper.lnrJT~'berc Lung Dis 1997;1: 4-93-4-97,6 Par~er SF, Cuoin WO. fne JSl" 0" tre dr'l"d 0000 SpOt sarrp e ir ep'cem c-Ior,;;c~suoi~. JClin Parrol 1999; 52(9): 633·639.7. CassOI S, Se as T, Gi MJ, t'fo. Stab' ity of or'ed 0oad Spot s:Jt"cimens 'or de:ec: on0" hUr"'a" :mmClrocefic"erC', vir ~s DNA by pOyrre'~se c"air rt"action. J GinM;crob;oI1992' 30: 3039-3042.2. :we:s F~sr e""jk~ M, Papo~'oeroJ M, /:rol. Stab ,it) or m.Man i~murooe"" ciercy:rus :'lpe 1~rt;ooc'es r .\rO e 0000 oried on fi:er pape' ana stored Jndel valouscoroifors :n K:rsrasa, la re. J(';n M'crco'CI19S2; 30: 1179-11829. 5aroa'" JAJ, Sa ~I'r R, C"l" s P, Con:re'as \1. Ge~: t1 pan'c ~ ~gg u::ra:'Ofl ass",', forHIV "n:'oOO es: rap'o, ecororr'c~1 moaification 'I/tn :rcreaseo sensiti~':Y. Mea Lcc'Sri 1998; 46: 135-1.:-0.

The use of dried blood spots and a modified particle agglutination test for cost-effective sero-epidemiological HIV surveys

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The use of dried blood spots and a modified particle agglutination test for cost-effective sero-epidemiological HIV surveys

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