Challenges in Virological Diagnosis of HIV -1 Transmission from Sexual Abuse – HIV-1 Genetic Links are Mandatory

Authors


Correspondence

Sven Grützmeier, Department of Microbiology, Tumor and Cell biology, Karolinska Institutet, Stockholm, SE-117 77, Sweden.

E-mail: Sven.Grutzmeier@ki.se

Abstract

The purpose of this article is to set forth possible strategies and techniques of analysis to diagnose or identify the source of HIV transmission in victims of sexual abuse. Diagnosis of HIV-1 transmission from sexual abuse is complicated. Timely blood samples are important. The right to confidentiality of the HIV diagnosis may prevent sampling from the offender. Hideous rapes occur during war, which victimize many women. Women delivering a child, seeking an abortion, or having a miscarriage may include victims of sexual abuse. HIV-infected children, where vertical transmission has been excluded, are important for investigation. Men who have sex with men may abuse young men. HIV-infected teenagers with signs of early infection should also be considered. Hundreds of single HIV-1 sequences can be created from one or more blood samples from the case and the alleged abuser. The more HIV-1 genes and sequences that are included, the better the outcomes of the phylogenetic relation. Evidence in support of transmission may be obtained from phylogenetic tree analysis and may also free someone from suspicion.

Introduction

At a time when most transmission routes of HIV-1 have been explored, HIV-1 transmission in connection with sexual abuse remains elusive and difficult to target. The purpose of this article is to set forth possible strategies and techniques of analysis to diagnose or identify the source of HIV transmission in victims of sexual abuse.

Strategies for sample collection and preservation

Target Groups

The following categories of cases may be important to keep in mind. Women calling for investigation of sexual abuse are the most obvious. Women delivering a child may include some victims of sexual abuse. This may also be true of women seeking an abortion and women having a miscarriage. HIV-infected children of HIV-uninfected mothers where there has been no blood transfusion or other similar risks are important for investigation[1] as well as HIV infection in children of HIV-infected mothers, where maternal transmission has been excluded. HIV-infected teenagers with signs of early infection should not be forgotten as possible victims of sexual abuse, including boys/young men who have sex with men. Finally, rape victims during war should be considered.

Ideally, a blood sample saved on a filter paper should be obtained from all abuse victims. Whenever the names of the possible offender(s) are identified a blood sample should be collected and be compared with the samples of the women. Even if a number of years may have passed, the HIV pattern may single out possible offenders. Here the relatively large number of possible offenders may facilitate identification of one or a few offenders.

Samples

If samples of the case and the alleged offender can be obtained in a reasonable time period after the suspected abuse, a careful analysis of the HIV-1 strains present in the abused and the suspect can point to a likely transmission source or eliminate a possible transmission source. However, earlier as well as later samples also can pinpoint a likely transmitted variant between two people.

In virological diagnosis of transmission, timing of sampling is everything. Samples should be collected as soon as possible at the time of suspicion of sexual abuse. Historical samples, if available, should be used also. The storage of historical samples and access to them are important. Ethical permission for taking and keeping samples must be respected. Legal issues may facilitate sampling but could also pose some barriers.

Samples from a control subject or control group are desirable. This may be easier said than done, however. In a research setting, controls could be required but still difficult to obtain. Contact tracing is important, but must respect ethical guidelines. Here HIV-infected subjects may be detected, where the link between the offender and victim may be challenged. In women, all possible contacts should be investigated. In intravenous drug users, it might be possible to obtain blood samples from others in the same network but identifying possible offenders outside this group may be more difficult. In abused children and teenagers, family contacts should be tested for HIV-1 antibodies. The widespread lack of knowledge requires research to be carried out from many areas of expertise, including legal aspects.

Sample Collection and Storage

There is more or less universal agreement on taking a blood sample from pregnant women for HIV-1. In those infected, their children also will be followed with samples until transmission can be ruled out. Such samples could preferably be stored. To reduce costs of keeping blood samples in a freezer, samples can easily be stored on filter paper and kept in room temperature in an inexpensive way and be useful for later diagnostic issues.

Filter paper storage of samples from women with abortions, whether spontaneous or induced, should be considered. Filter paper storage of samples from children born to HIV-infected mothers should be encouraged. Historical samples may prove pivotal for correctly diagnosing HIV infection by sexual abuse. Blood saved on filter paper can be used for analysis both of HIV-1 DNA, integrated in peripheral blood mononuclear cells(PBMC), and of HIV-1 RNA from HIV-1 present in plasma.

Otherwise, samples from plasma to detect HIV-1 RNA must be stored in at least −20°C, but best at −70°C. Similarly, HIV-1 DNA can be extracted from peripheral (PBMC) selected by centrifugation in a solution, which will eliminate other cells, such as red blood cells and polymorphonuclear cells. Then the cells need to be counted and similarly stored at −20°C, but best at −70°C. So, the filter paper technique may be the cheapest and most suitable for longtime storage.

Strategies for analyzing the HIV-1 genome

Polymerase Chain Reaction (PCR) and Sequencing PCR to Characterize Details in the HIV-1 Genome

In addition to the HIV-1 serological diagnosis, samples from HIV-infected cases can be investigated for their genetic similarity by sequencing different genes of HIV-1 or differences of HIV strains in cases and suspects by different PCR techniques. This procedure can be used both to eliminate suspicion and to support a possible or likely transmission chain.

Samples are prepared to get either pure DNA from cells or RNA from plasma. RNA must be transcribed to its DNA counterpart, as sequencing PCR is performed on DNA. For characterization of the DNA sequence, which codes for the respective protein of interest, the sample needs to be subject to nucleotide sequencing PCR. This can be obtained by doing one reaction per sample. This is often called population PCR. This provides the likely nucleotide in any given position.

To detect the numerous different HIV-1 strains present in blood, they need to be singled out. This can be done in different ways. We use a technique of limiting dilution PCR where a quadruplet set of fourfold dilutions is tested in PCR. This gives us the possibility to determine the best dilution, with which a high likelihood would yield single HIV-1 molecules. About 50–80 PCRs are tested at that dilution. When the outcome is about 25% positive reactions, these are likely to come from one single molecule. Whether this is so or not can be detected in the ensuing sequencing PCR of all PCR-positive samples[2, 3]. A single genome PCR is characterized by a unique sequence without any ambiguities. It can then be regarded as a single molecular clone. Newer techniques, such as pyrosequencing[4, 5], may increase the number of sequences available for genetic comparisons considerably.

Phylogenetic Tree Analysis of Related HIV-1 Sequences

When there are a number of sequences from the same gene segment, these can be compared in a phylogenetic tree, using different methods. We have used tree diagrams, which were plotted with FigTree v1.1.2, available at [http://mac.softpedia.com/get/Graphics/FigTree.shtml]. What is called ‘bootstrap values’ describes the likelihood that the different clusters in a tree mimic the actual genetic relationship. Values over approximately 90% display a high likelihood of genetic closeness.

Strategies for diagnosing HIV-1 transmission in sexual abuse

Table 1 illustrates the basic diagnostic parameters for a successful determination of HIV-1 transmission. Failure to obtain the information or samples of any of these components can jeopardize a correct diagnosis. Still, acquisition of many HIV-1 clones from single samples from the victim or suspect can be helpful. It may be assumed that it will be easier to obtain information and samples from the abused person than from the suspect.

Table 1. Virological analysis of HIV transmission in sexual abuse
 Samples in relation to the abuse
 HIV-1 testingPrior to the abuseaAt the time of the abuseLater samplesbPhylogenetic relationship
  1. a

    In seropositive cases, a relationship between the HIV-1 strains of the case and the suspect maybe more difficult to establish, but analysis using phylogenetic trees may still add valuable information.

  2. b

    There is a wide range in the temporal appearance of antibodies to HIV-1 after exposure. The shortest time of appearance of antibodies to HIV-1 is at 1 week after exposure. At 1 month, most infected would be seropositive, but some seroconvert after this time.

 

HIV-1 infection

proven at the

time of trial

Antibodies to HIV-1 absent

If negative but later positive supports

HIV-1 infection

at the time of the abuse

Negative up to

≈ 1 week post-exposure. Positive after ≈ 1 month

Phylogenetic link of

HIV-1 sequences from

the case and the suspect

with a high bootstrap

value

Suspect

HIV-1 infection

proven at the time

of trial

Antibodies to HIV-1 present

or absent

Antibodies to

HIV-1 present

Antibodies to

HIV-1 present

Phylogenetic tree analysis is a powerful tool to describe the genetic relatedness between sequences of the same part of a genome from different individuals. The analysis should preferably contain sequences with a low rate and those with a higher rate of mutations, for instance p17gag[6] and the V3 region of the envelope gene, gp120[2, 3, 7]. It should also contain reference sequences of the relevant subtype(s) and unrelated sequences from the same geographic area.

The time period after a rape that can be relevant for diagnosis is unknown, however, likely to remain for some or several years[2, 8, 9]. If all available samples are investigated, we will learn more about how and when the link may fade away. If the link between HIV-1 in the victim and the suspect remains and confirms other signs, it may assist in the diagnosis, but the absence of signs of a link cannot at present rule out a previous transmission if the only sample was obtained late.

Identification of the Relatedness of HIV-1 Strains in Mother-to-Child Transmission

Mother-to-child transmission provides an opportunity to study and determine the actually transmitted virus from mother to child. Here, there is no question that the mother is the source of the infection. We, and others, have studied transmission from mother to child using phylogenetic tree analysis of molecular clones[2, 3, 7]. Similarly, in sexual transmissions, the results of phylogenetic trees analysis may facilitate establishing a genetic link of HIV-1 between the victim and the offender.

The outcome of such phylogenetic trees of sequences from the mother and her child from different time points including clone sequences and sequences from viral isolates is shown in Fig. 1a–c, obtained from Clevestig et al. ([2]). This study focused on two coreceptor use phenotypes, R5 and X4, which are not part of this review and can be dismissed here. Only one part of the genome (gp120 V3) was used.

Figure 1.

(a) (left). Clonal expansion of HIV strains provides a high resolution of the genetic links of HIV-1. The phylogenetic trees were reproduced from Clevestig et al. ([2]) with permission from the publisher. Maternal sequences are labeled M, and child sequences C. Virus isolates, representing infectious virus, are either labeled di. (isolates from PBMC) or pi. (isolates from plasma). Clones end with a D for their PBMC origin. The numbers 1–4 of the child sequences represent the consecutive time points of the samples. (b) (right). The genetic links of HIV-1 between mother and child can be identified even years after the time of transmission. The child sequences denoted 2 are from 3 years of age. (c) Here all the child's sequences have been included in the phylogenetic tree. Despite the changes in HIV-1 sequences over time in the child, the links to the maternal sequences are still clear at the age of 5 years (labeled 4).

Figure 1a (left) shows the maternal sequences and the first sample from the child. It was easy to recognize the most likely transmitted virus, which was represented by a single maternal sequence, M3.R5.pi, from an infectious virus isolated from plasma at delivery. The child was HIV-1 negative at birth and therefore expected to have been infected during birth.

In Figure 1b (right), we see the relationship between the maternal sequences and sequences from the child, obtained at a later time point. The phylogenetic relatedness can still be traced. Here the isolate M3.R5.pi is still closest to the sequences from the child. Two clone sequences, M2.37D and M2.38D, are also close.

Figure 1c includes all samples from the child, including those obtained at 5 years of age when the viral population has mutated considerably. We can still observe the close connection between the maternal sequences M2.37D and M2.38D and the HIV-1 sequences in the child.

If we may use the mother and child as an analogous situation, where the mother represents the suspected ‘villain’ and the child represents the ‘victim’, then it becomes obvious that both later and earlier samples can be used to improve a diagnosis of HIV-1 relatedness.

Pinpointing the Relatedness of HIV-1 Strains in the Victim and the Suspect

Phylogenetic tree analysis has been used to trace sexual HIV-1 transmission links[8, 10-12]. Albert et al. ([10]) used phylogenetic tree analysis in a court case concerning rape. They sequenced HIV-1 pol and gag genes from both the victim and the suspect, including other HIV-1-infected cases. The conclusion was that the suspect most likely had transmitted the virus to the victim. In another study[11], a comparison of Bayesian and maximum-likelihood phylogenetic approaches was performed in two legal cases involving accusations of transmission of HIV, where both methods were informative. Leitner et al. ([8]) reconstructed a known HIV-1 transmission history by phylogenetic tree analysis. Campbell et al. ([12]) studied HIV-1 transmissions between discordant couples and identified about 70% linked transmissions, using new techniques for the determination of a linked transmission event.

Determination of the Direction of HIV-1 Transmission

It may be difficult in some cases to determine a direct transmission between the suspect and the victim and thus rule out another infected subject, carrying HIV-1 to the victim, as well as the direction of spread[9]. However, in a complicated epidemiological setting among three homosexual men, it was possible to deduce who had infected whom (direction)[13]. When children are involved, the direction should not pose a problem in most cases. Thus, the results from the phylogenetic analyses, the sequence distances between the virus from a man and the virus from a child, and the identification of the unique molecular fingerprint in the env gene, together indicated that the virus from the man and the virus from the child were epidemiologically linked[14]. Similarly, in suspicion of transmission from a dentist to a client, it could be shown that a blood transfusion was the cause of the subject's infection, not the dentist[6].

If the victim contracts a first HIV-1 infection from rape, the direction from the suspect to the case should be obvious. Also, if it can be established that the victim had not had sexual relations with any other person during the time of getting an HIV-1 infection, the conclusion of a direct (person to person) spread might be allowed. If intercourse had taken place with another subject, testing of this person may show whether there had been an exchange of any other HIV-1 strain. If not, this would lend support to the conclusion of a direct HIV-1 link between the case and the suspect.

Additional Considerations

There is great similarity between HIV-1 strains in recent transmissions. Such similarity may still exist in less timely samples but is expected to be less strong. However, it is possible to trace the origin of HIV-1 infection when samples are analyzed from the victim, the suspect, and unrelated cases. In cases of sexual abuse, it is recommended to use as many genetic sources (samples and genes) of HIV-1 as possible.

However, a proof of HIV-1 transmission does not prove that there has been a rape. Similarly, if rape has occurred and the suspect and victim are both HIV-1 infected, this alone does not prove that HIV-1 transmission was related to the rape. Still, sample collection and phylogenetic analysis should be encouraged, as it may add information, if not fully prove, a direct link between the suspect and the abused victim.

Conclusion

We have achieved many goals that seemed unrealistic decades ago. Research and altered attitudes now make it possible to investigate HIV transmission in cases of sexual abuse in women, children and men. Let us now promote sample storage of vulnerable cases, to allow for the necessary virological investigations.

Legal enforcement of laws to protect the victim's rights must be developed further. It is my conviction that the rights of the victim to identify a suspect must be given greater legal priority than the confidentiality of HIV-1-infection in a suspect. At present, this does not seem to be the case in many countries.

Acknowledgments

Financial support was given by the Karolinska Institute Research Funds, the Swedish Research Foundation, the Sven Jerring Foundation, SIDA (the Swedish International Development Agency). I thank Paul Muther for reading the manuscript. I am grateful for the possibility to use material published by Clevestig P, Maljkovic I, Casper C, Carlenor E, Lindgren S, Navér L, Bohlin AB, Fenyö EM, Leitner T, Ehrnst A in AIDS Res Hum Retroviruses, 21:371 (May); 2005.

Disclosures

The author has no potential conflicts of interest to declare.

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