[pronut-hiv] From NAM/Aidsmap:Early infant HIV diagnosis - part 1

["ProNut-HIV" <pronut-hiv@healthnet.org>]

>From NAM/AIDSMap

Early infant HIV diagnosis  
 

By Theo Smart

This HATIP reviews discussions at the 2007 Implementersâ Meeting and at the International AIDS Society (IAS) meeting a month later on early infant diagnosis, as well as some of the recent medical literature touching on the subject.

With thanks to:

Dr. Gayle Sherman, Department of Molecular Medicine and Haematology, National Health Laboratory Service and University of the Witwatersrand, Johannesburg, South Africa

Dr Tracy Creek, Centers for Disease Control and Prevention Global AIDS Program Prevention of Mother to Child Transmission Team

 
Key points
The first and best way to promote good health outcomes in HIV-exposed children is to make certain that every pregnant woman at risk of HIV receives a diagnosis. 

-                     If she is negative, she needs counselling and support to keep her that way since a woman is at high risk of infection during and just after pregnancy â and if infected, she is much more likely to transmit HIV to her baby.

-     If she is positive, she should receive:

-     the best care and treatment for her own health (including antiretroviral therapy (ART) for those who need it);

-    if she does not need ART for her own health, an optimal regimen to prevent-mother-to-child transmission (PMTCT) should be prescribed. 

Universal access to these HIV testing, counselling, care and prevention services should keep the vast majority of HIV-exposed children uninfected. 

In order to provide universal access, these HIV-related interventions must be more closely integrated with strengthened antenatal and other maternal child health (MCH) services to deliver the essential healthcare services needed by all mothers and infants in resource-limited settings. 

But at present, there are more than 2 million children with HIV, most of whom did not receive adequate PMTCT in time, and many of these children and their mothers remain undiagnosed.

Initiating ART in HIV-infected infants as early as 6-8 weeks (as soon as their infection status can be determined) dramatically reduces their risk of early death. In addition, identification and treatment of HIV-infected mothers can improve the survival of both the mother and her children.

Early infant diagnosis presents logistical challenges and is costly but possible

-     Antibody tests are unreliable in young infants while they carry their motherâs antibodies (out to 18 months though most infants will lose maternal antibodies long before that. WHO recommends rapid testing for infants >9 months old, followed by PCR only for those who are still antibody-positive.

-     Drawing, processing and shipping liquid blood samples from babies for PCR testing is impractical.

However, dried blood spots (DBS) can easily be collected from infants, packaged and sent to a centralised lab for reliable HIV testing by DNA PCR (see box 1 & 2 for procedure) 

HIV DNA PCR requires somewhat different equipment and techniques from those presently available in the labs scaling up viral load (HIV RNA) capacity in resource-limited settings â which raises the issue of how laboratory capacity should be allocated.

-    Running viral load tests on DBS has potential, but may not be as reliable or sensitive in infants whose mothers are on ART or who have received complex PMTCT regimens. Nevertheless, there would be significant advantages to using the same technology which is already being rolled out in many settings, so further research is urgently needed to validate the clinical utility of performing viral load on DBS samples from ART or PMTCT-exposed infants.

-     Other technologies are in development, but a point-of care test which could provide results on the spot seems a long way off.

For now, HIV DNA PCR on DBS is the gold standard for early infant diagnosis, but since this capacity will be very limited in most countries, the pilot programmes described in this article focus on setting up reliaised labs and to return the results as quickly as possible and get those infants who are positive into care and on ART 

Testing remains expensive but using a rapid HIV antibody test (RHT) to screen out negative results could reduce the number of samples that need to be collected and sent for PCR (see algorithm in article).

DBS should be performed on all HIV-exposed children, preferably at the time when they attend their first immunisation clinic.

HIV testing and counselling should be expanded in the mother-and-child health (MCH) systems of high HIV burden countries to identify all the mothers with HIV and HIV-exposed children, perhaps starting in the sick baby clinics since they will contain the most babies with HIV.

Community-based approaches are needed to expand access and acceptance of HIV testing and counselling for mothers and infants.

Adequate training, improved tracking of patients and their samples, timely transport of specimens, turn-around in the lab and delivery of results is crucial for DBS â otherwise, it is a waste of time.

For DBS to work, centralised referral laboratories will need to be strengthened to take on these new tasks and prevent backlogs. A long turn-around time could mean a useless result (and a childâs death).

Counselling has to be adapted to meet the challenges of giving parents what could be confusing test results.

There is a serious danger that mothers with HIV who receive an HIV-negative result on their infant will wean the child prematurely. The risk of early weaning and subsequent death of negative babies may be significant if the implications are not well understood by health providers and mothers.

Exclusive breastfeeding counselling must be reinforced, and programmes should consider ways of making breastfeeding safer, such as putting mothers at risk of transmitting HIV onto ART.

GET HIV-positive children quickly into care and treatment, otherwise testing is pointless

Newer, cheaper, faster tests for infants with HIV are urgently needed.
 

Putting systems in place for early infant diagnosis and treatment
Treating HIV-infected infants with antiretroviral therapy (ART) as soon as possible âwithin the first six to 12 weeks of life âreduces early mortality by 75%, according to the results from the Children with HIV Early Antiretroviral Therapy (CHER) trial presented at the 4th International AIDS Society Conference on HIV Treatment and Pathogenesis in Sydney Australia last year (Violari) (for a more complete review of the results, see http://www.aidsmap.com/en/news/973ABB13-4482-4A06-92BC-CC3298E9EF6E.asp).

Since more than half of the children who are HIV-infected may die before reaching two years of age without ART, and the median age of death in those who die early is around 6 months (Newell), these findings provide an obvious incentive, if not a moral obligation, to locate HIV-exposed infants, test them for HIV infection as soon as possible, and to get these children and their families into care and/or onto treatment.

Early infant diagnosis can serve as the bridge between prevention, care and treatment â and gauge the effectiveness of programmes for the prevention of mother to child transmission (PMTCT). But until recently, learning an infantâs HIV status at an early age has not been seen as financially or logistically feasible in most resource-limited settings. Recent programmatic experience with systems incorporating the collection of dried blood spots (DBS), which can be easily performed at even the most remote clinics and sent to a centralised laboratory for HIV testing (by PCR), may change all that.

âDBS is a simple procedure â as simple as doing a malaria slide â itâs not very difficult,â said Dr. Mildred Mudany of the CDC in Kenya at last yearâs HIV Implementerâs Meeting in Kigali, Rwanda, where reports about the implementation of DBS/HIV PCR systems for early infant diagnosis were made by nine PEPFAR-focus countries.

However, while the procedure itself is fa own set of operational challenges â especially in contexts where antenatal care, prevention of mother to child transmission programmes, and maternal and child health systems are not well-integrated, other basic maternal-child health interventions are not being implemented, reference laboratories are poorly equipped and staffed, systems for the timely delivery of samples and results arenât established, and linkages to HIV care and treatment are weak. 

These services must be strengthened in tandem and supported by community-based care systems for better case-detection and follow-up âto make certain that HIV-exposed children (and their mothers) don't fall through the cracks and do get linked into care.


Background on HIV testing in infants
Conventional antibody testing cannot reliably detect HIV infection in young children, since maternal antibodies to HIV can be passed onto the infant through the womb or through breast milk. These maternal antibodies may continue to be detected in uninfected infants out to 18 months using standard ELISA tests, though most infants will lose maternal antibodies long before that. WHO recommends rapid testing for infants >9 months, followed by PCR only for those who are still antibody-positive (see http://www.aidsmap.com/en/news/DAEE0A6A-109A-4D04-81C8-D88E111E0991.asp and more on this below). Nevertheless, study after study report the majority of HIV-exposed children are lost to follow-up long before that â and that, if infants cannot be tested earlier, the opportunity to get them into care and treatment could be lost.

Directly testing for HIV using PCR, on the other hand, is accurate when performed within 6 weeks of the last exposure (Dunn). But the test itself is expensive and can only be conducted at very well equipped laboratories by highly trained staff. But perhaps the greatest problem has been the logistics at the local clinic level: drawing adequate liquid blood samples from infants is difficult for non-specialists, and the proper processing, storage and transporting of so many liquid samples is simply too complex in many remote resource-limited settings.
 

On the proper collection, drying and packaging of DBS
Materials from Botswana

The following is text derived from two training materials posters put together by Tracy Creek and colleagues at the CDC for the Francistown pilot study 

Collection of Dried Blood Samples from Infants for PCR testing

1. Gather necessary supplies

-                     Gloves

-                     Blood collection card (filter paper)

-                     Lancet (2mm) (the Botswana study used a self-springing lancet)

-                     70% spirit or alcohol

-                     Gauze or cotton wool

-                     A pen 

2. Complete all necessary paperwork

-                    Infant diagnosis registration form
-                     Clinic register
-                     Laboratory request/report form

-                     DBS card 

3. Choose the area to be pricked and ask the mother to warm this area

-                     Infants 6 weeks-4 months: heel

-                     Infants 4 months -10 month: big toe

-                     Infants >10 months or >10 kg: finger 

4. Wash and glove hands. If gloves have powder, wash off powder 

5. Position the baby with the foot or hand down, then clean the spot to be pricked with spirit or alcohol, and allow to dry for 30 seconds 

6. Gently squeeze and release the area to be pricked until it is ready to be bled, then prick the infant in the selected spot with the 2mm lancet 

7. Wipe away the first spot of blood, then allow a large drop of blood to collect 

8. Touch the filter paper gently against the large drop and allow it to completely fill the circle. You may add new drops on top of wet drops if the circle is not full. Collect at least 3 good circles. Do not put a fresh drop on top of one that has already dried. 

9. Clean area, no bandage is needed. 

Drying and P area for at least 4 hours or overnight.

2. Keep lab request forms with DBS cards 

Packaging

1. Wrap the individual DBS card with a glassine paper so that DBS cards will not have direct contact with each other. Insert up to 10 wrapped cards into a special sealable plastic bag.

2. Add 10 desiccant packets to each bag. 

3 Add at least one humidity card per bag. Gently press the bag to remove most of the air before sealing. 

4. Use the specimen delivery checklist to check you have a lab form for each DBS.

- Place the bag of DBS, all the DBS DNA PCR lab forms and the specimen delivery checklist into a large envelope.

-  Label the envelope with:

o Name of collection site (clinic)

o Name of person delivering specimen

o Date you are sending the samples

- Place the envelope in designated area to be picked up for the laboratory

For copies of these two posters (which contain full color pictures illustrating the procedure) and other related CDC materials, please contact Dr Tracy Creek at tgc0@CDC.GOV

Materials from the International Center for AIDS Care and Treatment Programs (ICAP)

ICAP has also developed an extensive library of paediatric HIV/AIDS resources for clinicians in resource-limited settings: see http://www.columbia-icap.org/resources/peds/index.html

One of these is a clinical manual on infant diagnosis of HIV:

http://www.columbia-icap.org/resources/peds/files/Infantdx050307.pdf

ICAP has also put together a ppt presentation, which also provides a detailed step by step visual demonstration on proper DBS collection, drying and packaging:http://www.columbia-icap.org/resources/peds/files/Module7-Collection,storage%20and%20transportation%20of%20DBS-2007.ppt

(This is a large file, at approximately 10 MB)
 

DBS testing: accurate and more practical
âDried blood spots (DBS) can overcome the blood sampling and logistical obstacles that limit access to infant diagnosis in low-resource settings,â Dr Gayle Sherman and colleagues in Johannesburg wrote in JAIDS in 2005. Their prospective cohort study comparing PCR testing on DBS versus liquid blood specimens from 288 infants at the age of 6 weeks, yielded an accurate diagnosis of HIV infection status, with only one false positive, which was detected on repeat testing. 

A large-scale evaluation project, by Dr Tracy Creek and colleagues in Botswana, successfully piloted the use of DBS HIV PCR as part of routine infant care in a public health system. A preliminary account of Dr Creekâs results were first presented at the 2006 HIV Implementers meeting in Durban, South Africa, but has since been published in the January 1st 2008 issue of the Pediatric Infectious Disease Journal. 

This joint project between Botswana and the CDC developed new policies, procedures, and training materials to introduce a DBS testing system into 15 clinics and one referral hospital in Francistown, and the Botswana-Baylor Childrenâs Center of Excellence Clinic in Gaborone. 

The collection, drying and packaging procedures were relatively simple. Drops of blood from each infant were taken from a toe or heel prick by the local clinic staff on filter paper, then dried, packed with desiccant and stored and/or sent to the centralised laboratory (For a more detailed description of the procedure, and related resources, see boxes 1 and 2).

Training on the procedure consisted of a one-day classroom session given to clinic staff, followed by a few days practical supervised training on-site. Then, over a six-month period between June and December 2005, staff collected and shipped specimens from 1931 HIV-exposed infants to a centralised laboratory for testing. The laboratory only rejected 27 (1.4%) samples â mostly due to labelling errors. 136 (7.0%) of all the infants were HIV-infected and entered into care. Retesting of 150 random samples for quality assurance at US Centers for Disease Control (CDC) in Atlanta showed 100% concordance with the tests performed in Botswana. 

After this successfationwide during 2006â2007 with the goal of providing âearly diagnostic services to the approximately 13,300 HIV-exposed infants born in Botswana each yearâ (more on the challenges scaling up to that level in Botswana is reported below).

Further experiences using DBS for PCR testing in Rwanda, Mozambique and South Africa were then presented at the World AIDS Conference in Toronto in 2006.

Dr Luis Manuel Felipe Gonzalez, who works as laboratory adviser for the International Center for AIDS Care and Treatment Programs (ICAP) in Eastern Africa, reported on the introduction of DBS in Rwanda â and described how the logistics (transport, follow-up, etc,) were adapted somewhat to meet the local needs and resources in that environment (this will be discussed in more detail below). 

The lab and sites Dr Gonzalez set up also served as centres of excellence, training those interested in introducing similar programmes into neighbouring countries. 

Mozambique also piloted a project, reporting that DBS â allowed the diagnosis of children from remote areasâ and serves as a tool for treatment and care entry, establishment of follow-up programs for both HIV-positive and breast-feeding HIV-negative infants, implementation of guidelines for counselling on infant feeding, and allows treatment of exposed children in a timely manner.â (BilaRamalho).

Dr Nigel Rollins of the University of KwaZulu-Natal, reported on the use of DBS in a pilot programme set up to perform routine anonymous HIV surveillance testing on all infants aged between four to eight weeks who were brought in for their first DTP immunisation at seven primary health care clinics offering PMTCT services in KwaZulu Natal (KZN). 

The protocol was different: DBS were collected from 2,439 infants between the ages of 4-8 weeks and first screened for antibodies to determine whether the infant was HIV-exposed, and then, the 907 samples that were antibody-positive were tested for HIV by PCR. The study detected a vertical transmission rate of 20.8% in 6-week old infants of HIV-positive mothers â and served as a clear indication that the PMTCT programme in that province was not reaching its targets (for more on this study, see http://www.aidsmap.com/en/news/74A5B372-0FAF-4EB1-8825-F84DDAB7D367.asp).

Dr. Rollins said the procedure âhas the added advantage not only of identifying children early for treatment â getting them and the mother as well into the continuum of care, but it is a very effective way of monitoring the PMTCT system.â