Discordant HIV DNA PCR results among infants diagnosed with HIV infection and initiated on ART: a case series

Abstract

This case series reports three infants diagnosed with HIV-1 infection using DNA polymerase chain reaction (PCR) testing. The three children were initiated on antiretroviral therapy (ART) at ten, four and six months of age. Their serological tests at 18 months of age were negative for HIV-1. The first child was discontinued from ART. The other two children were HIV-negative after 18 months, but were continued on ART. Such seroreversion may be either due to viral suppression or false-positive DNA PCR results. There is a need to develop guidelines to address such discordant cases.

Keywords

Early infant diagnosis EID HIV-1 DNA PCR DBS regional research laboratories integrated counseling and testing centres

Background

Perinatally-acquired HIV infection is associated with rapid progression in infants.^1,2 In the absence of treatment, more than a third of HIV-infected infants die during infancy.^3,4 The Children with HIV Early Antiretroviral Therapy study demonstrated the benefits of early initiation of antiretroviral therapy (ART). In view of this evidence, the World Health Organization (WHO) recommended early infant diagnosis (EID) of HIV infection.⁵ Since 2010, EID services have been provided by the National AIDS Control Programme (NACP) in India.⁶

India’s EID programme involves testing of dried blood spot (DBS) specimens from HIV-exposed children at six weeks of age by HIV-1 DNA polymerase chain reaction (PCR) test (Roche Amplicor) (GOI 2013).⁷ A whole blood (WB) sample is collected for repeat HIV-1 DNA PCR testing from those infants whose DBS samples show presence of HIV-1. A third blood sample (WB) is collected if the results of the first two tests are discordant. The EID tests are conducted at seven Regional Reference Laboratories (RRLs). At 18 months of age, all HIV-exposed children are tested at integrated counseling and testing centres (ICTCs) for the presence of HIV antibody. We report three children diagnosed with HIV-1 infection in the EID programme and initiated on ART. All three had negative HIV test results at 18 months and thereafter.

The cases

The three cases presented in this paper were detected positive for presence of HIV-1 with the DNA PCR test on DBS samples at the National AIDS Research Institute, despite them receiving a single dose of nevirapine immediately after birth. Details of these cases are provided in Tables 1 and 2. WB samples were obtained from these children for confirmation of diagnosis. In one case, the first WB sample was negative but the second WB sample tested positive for HIV-1, and diagnosis was thus confirmed. In the other two cases, the first WB sample was positive, confirming diagnosis.

Table 1.

Profiles of the three HIV-exposed infants.

Characteristics	Child 1	Child 2	Child 3
Time of diagnosis of mother	Fourth month of pregnancy	Third month of pregnancy	HIV-positive prior to pregnancy
Was mother started on ART?	Yes (AZT+3TC+NVP)	No	Yes (AZT+3TC+NVP)
Type of delivery	Elective caesarian section	Full-term normal delivery	Full-term normal delivery
Sex	Male	Male	Female
Baby received NVP at birth	Yes	Yes	Yes
Time of initiation of ART	10th month	Fourth month	Sixth month
ART combination	AZT+3TC+LPV	AZT+3TC+LPV	ABC+3TC+LPV
Baby CD4 cell count at diagnosis	1170/mm³ (44%)	1236/mm³ (31%)	2130/mm³ (42%)

AZT: zidovudine; 3TC: lamivudine; NVP: nevirapine; ART: antiretroviral therapy; ABC: Abacavir.

Table 2.

Diagnostic details of the three HIV-exposed infants.

Characteristics	Child 1	Child 2	Child 3
Time of collection of the first DBS sample	Third month	Six weeks	Six weeks
DNA PCR result of DBS sample at RRL	HIV-1-positive	HIV-1-positive	HIV-1-positive
Time of collection of second (whole blood) sample	Fifth month	Third month	Sixth month
DNA PCR result of second test at RRL	HIV-1-negative	HIV-1-positive	HIV-1-positive
Time of collection of third (whole blood) sample	Ninth month	–	–
DNA PCR result of third test at RRL	HIV-1-positive	–	–
Results of antibody tests at 18 months at ICTC (Comb AIDS, Tri-line and Trispot)	All three negative	All three negative	All three negative
Results of repeat antibody tests at ICTC	All three negative at 43rd month	All three negative at 19th month	All three negative at 18th month
Results of DNA PCR tests at RRL	Not done	Negative at 23rd month	Negative at 19th month

ICTC: integrated counselling and testing centre; RRL: regional research laboratory; DBS: Dried blood spot.

These three infants were initiated on ART at ten, four and six months of age, respectively. Their serological samples were collected at 18 months of age as per NACP protocol. All three children were found to be negative with three rapid serological diagnostic tests (Comb AIDS- Span Diagnostics, Surat, India; Tri-line- Bhat-Biotech, Bangalore, India; Trispot- J. Mitra & Co., New Delhi), following NACO guidelines⁶ at the ICTC. The first child was discontinued from ART and a repeat antibody test at 43 months at the ICTC was negative. A follow-up DNA PCR test was not done for the first child. For the other two, DNA PCR tests at RRL were negative. Both these children were continued on ART. None of the children had ever had any sign or symptom suggestive of opportunistic infection. All three children are asymptomatic.

Discussion

All three cases reported in this paper had negative HIV test results while on ART. Do these three cases resemble the ‘Mississippi baby’? Does failure to detect virus indicate suppression of virus by ART? The Mississippi baby was initiated on ART very early, right after birth.⁸ In the first case, ART was initiated too late and was given for less than one year. Even in the other two children, ART was initiated only after three months of age. This was inadequate for achieving functional cure given current scientific evidence. In the Mississippi baby case, investigators reported absence of HIV-1 after extensive virological and immunological evaluation.⁹ While cases 2 and 3 were confirmed HIV-negative by two consecutive DNA PCR tests, the first case gave a negative result in a second DNA PCR test. However, the third test was positive. A more specific RNA-PCR test may be appropriate to confirm diagnosis in such cases. However, the programme had no provision for RNA-PCR testing. False-positive HIV-1 DNA PCR results, although not common, have been reported.^10–13 The three children reported here might be cases of false-positive DNA PCR during infancy. However, it is also possible that these children were infected with HIV and ART resulted in suppression of the virus. This could result in false-negative post-18-month rapid tests and HIV-1 DNA PCR tests. Such cases have been reported from Lesotho, Africa.¹⁴

Since every child was diagnosed with HIV-1 infection only after two positive HIV-1 DNA PCR tests and the RRL has robust quality control mechanisms as certified by National Accreditation Board for Testing and Calibration Laboratories, it is unlikely that the technical errors could have contributed to the findings.

These findings have implications for a large public health programme. Failure to detect HIV after initiation of ART may be due to viral suppression or false-positive DNA PCR tests during infancy. It is important to differentiate between the two in order to decide a further course of management for children with such discordance. In the first scenario, interruption of ART may lead to reappearance of the virus as was seen in the Mississippi case and may result in clinical deterioration.⁹ In the second scenario, the child will unnecessarily be subjected to ART.

This report of three children and previous case reports from India^10–12 make it imperative to revisit the guidelines related to discordance of HIV results among exposed infants. Should the children with such discordant results be subjected to viral load tests and treatment stopped if negative? In light of the reappearance of HIV in the Mississippi baby after 27 months, withdrawal of treatment is debatable. There are no clear cut guidelines for discontinuing ART in such cases.¹⁵ There is a need for consolidation of available evidence and guidelines for the management of such cases. There are also the social and psychological implications of disclosing these results to the parents of a baby. Guidelines for counseling needs in such cases is essential.

Footnotes

Disclaimer

The views expressed in this paper are solely those of the authors.

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The authors acknowledge National AIDS Control Organisation (NACO) for providing support through Early Infant Diagnosis programme.

References

Violari

Cotton

Gibb

. for the CHER Study Team. Early antiretroviral therapy and mortality among HIV-infected infants. N Engl J Med 2008; 359: 2233–2244.

Bourne

Thompson

Brody

. Emergence of a peak in early infant mortality due to HIV/AIDS in South Africa. AIDS 2009; 23: 101–106.

Newell

Coovadia

Cortina-Borja

. the Ghent International AIDS Society (IAS) Working Group on HIV Infection in Women and Children. Mortality of infected and uninfected infants born to HIV-infected mothers in Africa: a pooled analysis. Lancet 2004; 364: 1236–1243.

Marinda

Humphrey

Iliff

. the ZVITAMBO Study Group. Child mortality according to maternal and infant HIV status in Zimbabwe. Pediatr Infect Dis J 2007; 26: 519–526.

WHO. WHO recommendations on the diagnosis of HIV infection in infants & children. Geneva: WHO, 2010.

Government of India, National AIDS Control Organization. National guidelines for prevention of parent-to-child transmission (PPTCT) of HIV, www.naco.gov.in/upload/Publication/Basic%20Services/National%20Guidelines%20for%20PPTCT_01_05_2013.pdf (2013, accessed 2 September 2016).

Government of India, Ministry of Health and Family Welfare, Department of AIDS Control. Updated guidelines for prevention of parent-to-child transmission (PPTCT) of HIV using multi drug anti-retroviral regimen in India, December 2013. Available at: http://www.naco.gov.in/documents/policy-guidelines (accessed 14 September 2016).

Persaud

Gay

Ziemniak

. Absence of detectable HIV-1 viremia after treatment cessation in an infant. N Engl J Med 2013; 369: 1828–1835.

Luzuriaga

Gay

Ziemniak

. Viremic relapse after HIV-1 remission in a perinatally infected child. N Engl J Med 2015; 372: 786–788.

10.

Mathai

Adhikari

. Repeated false positive HIV DNA PCR in an exposed infant. MJAFI 2013; 69: 392–393.

11.

Shah

. Efficacy of HIV PCR techniques to diagnose HIV in infants born to HIV infected mothers – an Indian perspective. J Assoc Physicians India 2006; 54: 197–199.

12.

Agarwal

Agrawal

. False positive HIV-1 DNA PCR in infancy. Indian Pediatr 2008; 45: 245–246.

13.

Baveewo

Kamya

Mayanja-Kizza

. Potential for false positive HIV test results with the serial rapid HIV testing algorithm. BMC Res Notes 2012; 5: 154–157.

14.

Garcia-Prats

Draper

Sanders

. False-negative post-18-month confirmatory HIV tests in HIV DNA PCR-positive children: a retrospective analysis. AIDS 2012; 26: 1927–1934.

15.

Volmink

Marais

. HIV: mother-to-child transmission. Clin Evid 2008; 2008: 0909–0909.