# Presentation: 142721

## D R Congo ANC Surveillance: 

- Piloting Dried Blood Spots (DBS) and HIV Rapid Test (Determine and Oraquick) in Two urban Sites (Bukavu and Kisangani)

- Presenter: Mr. Nicaise M. Agbala
- Medical Technologist, PNLS, HIV/STI National
- Reference Laboratory, Kinshasa, DRC

**Notes:**
I am pleased to present the DRC experience on the lab support to ANC surveillance, specially the pilot study on dried blood spot and rapid HIV tests in two town in the eastern DRC: Bukavu and Kisangani

##     Objectives:

-   To evaluate DBS as an alternative strategy
-     for  collecting samples for surveillance
-     in remote areas without cold chain.
-   To evaluate the performance of DBS as
-    QC for Rapid Test
-   To introduce and evaluate a second HIV
-     rapid test Oraquick on venous whole blood
-     in parallel with HIV Determine

**Notes:**
The three objectives of this study were:
First  to evaluate DBS as an alternative strategy  for  collecting samples for surveillance  in remote areas without cold chain given that the cold chain is a major problem in our country.

 second to evaluate the performance of DBS as  Quality Control  for Rapid Test used in surveillance, VCT, PMTCT

  third to introduce and evaluate a second HIV
    rapid test Oraquick on venous whole blood which can be stored at room temperature in parallel with HIV Determine usually used 

## Methods:

-  Pilot study during ANC surveillance
-    in Eastern DRC, Kisangani and Bukavu
-  Sampling in ANC/PMTCT women
-  Two-day training in surveillance operation
-    for interviewers and lab technicians from
-    collection sites and regional labs

**Notes:**
For that , 

A Pilot study was conducted in january 2004 during ANC surveillance   in Kisangani and Bukavu town.
 Interviewers and lab technicians from collection sites and regional labs were trained in surveillance operation for two days by central supervisors with CDC Atlanta experts and CDC/DRC technical assistance
Sampling was done among pregnant women attending  ANC and PMTCT services; acceptance rate was more than 99%.

##    Methods:

-   Following day, start of surveillance
-    activities and monitoring activities of
-    that first day
-   Two PNLS supervisors (Epi and Lab)
-     remained on site until end of sampling
-     period

**Notes:**
After the two-day training, surveillance activities started .

  Two PNLS supervisors (Epi and Lab)
    remained on site until the end of the sampling  period to monitor surveillance activities

##    Methods:

-   Gold standard algorithm for ANC
-    surveillance ( National protocol) is HIV
-   Determine in sera as a  screening test in
-   site laboratory,
-    then HIV Enzygnost anti-HIV 1/2 Plus (EIA test) performed at the PNLS Lab
-    as a  supplemental test
-    All discordant samples between Determine  and Enzygnost retested with both tests.

**Notes:**
According to National protocol on HIV surveillance, we use HIV Determine in sera as a  screening test in  site Laboratory,

Then, a  HIV EIA test is performed at the PNLS Lab  as a  supplemental test, in our protocol we use Enzygnost anti-HIV 1/2 Plus). 

Concordant results between Determine and Enzygnost are considered eitheir Negative or Positive for final serostatus

##    Methods:

-   For concordant results between Determine
-    and Enzygnost, final serostatus either
-    Negative or Positive
-   Final serostatus given by Determine result
-    for discordant results

**Notes:**
All discordant samples between Determine  and Enzygnost were retested with both tests.

 For discordant results, final serostatus is  given by PNLS Lab Determine result.

This is our  gold standard algorithm for HIV surveillance in ANC.

## Methods:

-  Only for Bukavu and Kisangani, DBS tested
-    with Vironostika HIV Uniform II Plus O at the
-    PNLS Lab, and HIV rapid test Oraquick
-    was added to the testing on sites.
-    Their results  were compared to the gold
-     standard algorithm
-  Results of HIV Determine performed on sites with whole blood  were compared to HIV EIA and Determine repeated at the PNLS Lab with sera to check on discordance between site testing and Reference Lab

**Notes:**
For  Bukavu and Kisangani, HIV Determine was performed on sites with whole blood.
A second HIV rapid test, Oraquick,  was added to the testing on sites.
 DBS was collected on site and tested  with Vironostika HIV Uniform II Plus O at the  PNLS Lab
At the  PNLS Lab , Enzygnost EIA was performed and we used Determine HIV rapid test  with sera to determine the final serostatus in case of discordance. 
Results of HIV Determine performed on sites were compared to the gold standard algorithm to check on discordance between site testing and Reference Lab

DBS performance was compared to the gold standard algorithm

## Methods:

-   Sensitivity, specificity, positive predictive
-    value and negative predictive value calculated
-    for DBS and Oraquick performance for each
-    site
-   The total numbers of concordant results on all
-    tests (Determine, Enzygnost, Oraquick, DBS,
-    and Serostatus) recorded as well as the
-    discordance on one test or another

**Notes:**
Performance characteristics for DBS and Oraquick tests were studied using  the calculation of  Sensitivity, specificity, positive predictive value and negative predictive value for each site

  The total numbers of concordant results on all
   tests (Determine, Enzygnost, Oraquick, DBS, 
   and Serostatus) were recorded as well as the
   discordance on one test or another

##     Methods:

-     Methods:
-    Paired DBS samples (HIV Vironostika
-    Uniform II Plus O) and plasma (Enzygnost
-     anti-HIV 1.2. Plus) retested in Atlanta as
-     part of external quality assessment to
-     check on overall quality of the results
-     given by the PNLS Lab, and discordance
-   Recommendations were made as lessons
-    learned from the pilot experience

**Notes:**
Paired DBS samples and plasma were sent to Atlanta  to be retested  as part of external quality assessment to check on overall quality of the results given by the PNLS Lab, and discordance.

  After that, recommendations were made as lessons learned from the pilot experience 

## Results:

**Table 1.      Performance of DBS compared to **
**                   ****gold standard algorithm**
-                         **Bukavu**        **Kisangani **
**                                ****n=513              n=511**
- Sensitivity               71.43%            67.74%
- Specificity               99.60%          100.00%
- Positive P. V.          83.33%          100.00%
- Negative P. V.         99.20%           97.96%
**    ****ORAQUICK results showed exactly the same performance as DBS in Bukavu and in Kisangani**

**Notes:**
Results:

Table 1.      Performance of DBS compared to 
                   gold standard algorithm
                              Bukavu        Kisangani 
                                n=513              n=511
Sensitivity               71.43%            67.74%                                  
Specificity               99.60%          100.00%
Positive P. V.          83.33%          100.00%    
Negative P. V.         99.20%           97.96%

    ORAQUICK results showed exactly the same performance as DBS in Bukavu and in Kisangani

## Results:

**Table 2. Concordance/discordance between tests   **
**              ****after completion of QC testing at PNLS**
-                                               **Bukavu            Kisangani**
**                                               ****n= 513               n=511**
-  Concordance in all tests         500                      498
-  simultaneously                      (97.47%)          (97.46%)
-  Discordance in one test          13                        13
-  or another                              (2.53%)            (2.54%)

**Notes:**
Table 2. Concordance/discordance between tests   
              after completion of QC testing at PNLS

                                              Bukavu            Kisangani
                                               n= 513               n=511

 Concordance in all tests         500                      498
 simultaneously                      (97.47%)          (97.46%)

Discordance in one test          13                        13
 or another                              (2.53%)            (2.54%)

##  Results:

- • Collection of DBS in remote areas without
-   cold chain is feasible and makes sense
-   logistically
- • Rapid tests with whole blood comparable
-  with serum
- • Some difficulty in reading HIV Determine
-  bands (shadow) for not so experienced eyes
-  created a confusion among testing staff,
-  increased false positive rates and discordan-
-  ce between sites testing and PNLS testing

**Notes:**
Collection of DBS in remote areas without 
  cold chain is feasible and makes sense
  logistically
• Results from rapid tests with whole blood are comparable to results from serum
• Some difficulty in reading HIV Determine 
 bands (shadow) for not so experienced eyes
 created a confusion among testing staff, leading to increased false positive rates and discordan-
 ce between sites testing and PNLS testing

## Results: EQA testing in Atlanta

-  At reception, the samples were very well packaged for the DBS, not so for plasma and sera. They were intact, in cold temperature
- DBS circles for Bukavu were well done, for Kisangani the blood from each circle touched the other because 110ul of anemic blood spreads too much
- • EQA testing for DBS and Plasma respectively with Vironostika HIV Uniform 1/ 2 Plus O and Enzygnost anti-HIV 1.2 Plus confirmed that the use of DBS is a suitable alternative strategy  of blood collection in logistically challenged areas and a good QC tool for rapid testing

**Notes:**
Results: EQA testing in Atlanta

 At reception, the samples were very well packaged for the DBS, not so for plasma and sera. They were intact, in cold temperature

DBS circles for Bukavu were well done, for Kisangani the blood from each circle touched the other because 110ul of anemic blood spreads too much  

• EQA testing for DBS and Plasma respectively with Vironostika HIV Uniform 1/ 2 Plus O and Enzygnost anti-HIV 1.2 Plus confirmed that the use of DBS is a suitable alternative strategy  of blood collection in logistically challenged areas and a good QC tool for rapid testing

## Results: EQA testing in Atlanta

- Type of mismatch   Bukavu     Atlanta      Total
- Serum Enzygnost               Negative        Low positive    3/513 (0.6%)
- DBS  ELISA                         Positive        Negative          2/513 (0.38%)
- DBS ELISA/ Enzyg.             Positive         Negative          2/513 (0.38%)
- Bukavu         Atlanta
- Determine/All other tests      Positive        Negative         5/513 (0.97%)

## Results: EQA testing in Atlanta

- Type of mismatch   Kisangani  Atlanta       Total
- Serum Enzygnost              Negative        Low positive    1/511 (0.6%)
- DBS  ELISA                         Negative        Positive         1/511 (0.38%)
- DBS ELISA/ Enzyg.             Negative         Positive          4/511 (0.78%)
- Bukavu         Atlanta
- Determine/All other tests      Positive        Negative         9/511 (0.97%)
- Overall good quality and reliable data from
-  the pilot testing. DBS can be used for surveil-
- lance with or without rapid test

## Recommendations/Lessons Learned

-  Timing for preparation
- • Anticipation delays in reception of goods
- • Sites assessment as soon as planning of survey
-    decided
- • Contacting and including regional labs and
-   collection sites directors at the start of planning
- 2. Assignment of responsibilities for different tasks
-    (e.g. purchase of goods, making labels, sites
-     assessment, ....)
- 3. DBS collection ideal; HIV rapid tests on site with
-      two tests in parallel and one different tie-breaker; DBS testing in regional lab should be encouraged

**Notes:**
Recommendations/Lessons Learned
1.Timing for preparation:
• Anticipation delays in reception of goods
• Sites assessment as soon as planning of survey  decided
• Contacting and including regional labs and 
  collection sites directors at the start of planning
2. Assignment of responsibilities for different tasks
   (e.g. purchase of goods, making labels, sites
    assessment, ....)
3. DBS collection ideal; HIV rapid tests on site with 
  two tests in parallel and one different tie-breaker; DBS testing in regional lab should be encouraged

## Recommendations/Lessons Learned

- • Minimum 3 days of training/Follow-up
- • Review National HIV testing algorithm
- • Training of Regional Labs in DBS
-    EIA for future surveillance
- • PNLS for EQA role only
- • Enrollment in CDC proficiency
-    testing in DBS
- • Capacity building of small labs to
-   support Surveillance/PMTCT/VCT activities

**Notes:**
Minimum 3 days of training/Follow-up
• Review National HIV testing algorithm
• Training of Regional Labs in DBS
   EIA for future surveillance
• PNLS for EQA role only
• Enrollment in CDC proficiency
   testing in DBS
• Capacity building of small labs to 
  support Surveillance/PMTCT/VCT activities

## Acknowledgement:

- • All Staff of HIV/STI National Reference
-   Lab, PNLS, Kinshasa, DRC
- • Dr. Jack Kokolomami, Director,PNLS
-   and his dedicated Epidemiology Staff
- • All Regional PNLS Directors
- • All Staff and Directors of Bukavu and
-   Kisangani Regional Hospital Laboratories
- • All Staff of Collection sites
- • CDC Director GAP-DRC and her dedicated Staff
- • CDC DASTLR HIV and STI Laboratory Support
-   Teams

## Bukavu General Hospital Lab

## C S  Funu Lab in Bukavu