We tested for HIV in discarded needles and syringe washes from 191 HIV-infected patients receiving injections in rural Cameroon. HIV-1 RNA was amplified from 34 of 103 intravenous injection syringes and two of 88 intramuscular injection syringes. All 36 strains were HIV-1 group M. The majority belonged to the circulating recombinant form CRF02 (IbNg). Our data support a role for unsafe injections in the spread of HIV-1 in Africa, in contrast to recent studies.
aTulane National Primate Research Center, Covington, Louisiana 70433, USA
bDepartment of Tropical Medicine, Tulane University School of Public Health and Tropical Medicine, New Orleans, Louisiana 70112, USA
cMedical Research Station, Kumba, Cameroon, USA
dTheoretical Biology and Biophysics, Group T-10, Los Alamos National Laboratory, Los Alamos, New Mexico 87545, USA
eAlbert Einstein College of Medicine, Bronx, New York, USA.
Received 10 November, 2005
Revised 2 February, 2006
Accepted 24 February, 2006
Sponsorship: This work was supported by funds from grant RO1 AI-44596.
HIV transmission through the reuse of needles and syringes may fuel the spread of HIV. Several epidemic outbreaks in eastern Europe [1,2] and Libya  have shown a rapid spread of HIV in nosocomial settings in the absence of standardized precautions. Moreover, transmission of HIV through contaminated needles and syringes was reported to contribute to the adaptation of cross-species transmitted SIVcpz and SIVsm to became the pathogenic HIV-1 and HIV-2, respectively [4,5]. A recent study carried out in Ethiopia failed to find HIV in used needle flushes . Moreover, another study excluded unsafe injections as a major source of HIV infection in Africa . Therefore, our aim was to investigate the risk of HIV transmission through unsafe injections using a more sensitive approach.
Matched blood and syringe washes were obtained from 191 HIV-infected patients receiving injections in 12 clinical settings in the southwest province of Kumba, Cameroon, in a 3-month period in 2001–2002. Written consent was obtained from all patients and the study was approved by the review board of the Medical Research Station, Kumba. The syringe washes were performed within 6 h of the syringe being used. The syringes were flushed with 1 ml RNA later (Ambion Inc., Austin, Texas, USA), which was drawn into the barrel and then flushed out through the needle. Blood specimens and syringe washes were preserved in ‘RNA later’. RNA and DNA were extracted from 500 μl of the syringe washes using a Qiagen QIAamp Viral RNA extraction kit (Qiagen, Valencia, California, USA). DNAses were not used, allowing the concomitant extraction of DNA. Control samples consisted of blood and syringe washes from 150 HIV-negative patients, tested under the same conditions. Reverse transcription (RT) polymerase chain reaction (PCR) or DNA PCR were performed on all samples using primer combinations targeting different structural genes as described . PCR products were directly purified using a QiaQuick gel extraction kit (Qiagen). Sequences were obtained with an ABI Prism Dye Terminator Cycle Sequencing Ready Reaction kit with AmpliTaq DNA polymerase (Perkin-Elmer, Wellesley, Massachusetts, USA) on an automated sequencer (Applied Biosystems 373A, Foster City, California, USA) and submitted to GenBank (accession numbers: DQ452626–DQ452686).
The gag, pol and env nucleotide sequence alignments were obtained from the Los Alamos National Laboratory HIV Sequence Database (http://hiv-web.lanl.gov). Newly derived sequences were aligned with the reference sequences using CLUSTAL W  and adjusted manually. Regions of ambiguous alignment and all gap-containing sites were excluded. Phylogenetic trees were inferred from the nucleotide alignments by the neighbor-joining method .
No samples from HIV-negative patients were positive. A relatively high number of PCR on specific gene fragments were obtained by testing syringe washes from HIV-1-infected patients (Table 1). At least one genomic fragment was amplified from 36 of 191 HIV-1 seropositive specimens. Thirty-three per cent (34 out of 103) of the syringes used for intravenous injections in HIV-seropositive individuals contained amplifiable virus. Only 2% (two out of 88) of the syringes used for intramuscular injections contained amplifiable virus. This is not surprising because the exposure to blood during intramuscular injections is significantly lower than in the case of intravenous injections. However, these results show that there is a risk of HIV transmission even in the case of intramuscular injections.
PCR results with different primer sets are shown in Table 1. The most sensitive PCR assay targeted the protease gene: 29 samples were positive in this region. Fifteen samples were positive in RT, 13 in env and 17 in gag. These differences in PCR efficacy were probably related to the first round PCR for RT, env and gag targeted larger amplicons.
In phylogenetic trees (data not shown), the majority of the gag sequences clustered with CRF02 (n = 12); the remaining ones clustered with subtypes D (n = 1), F2 (n = 1) and K (n = 1) and CRF11 (n = 1). Out of the 27 protease sequences, 19 clustered with CRF02, two with subtype D (n = 2), four with CRF01 and two with CRF11. Most of the RT sequences clustered with CRF02 (n = 10), the remaining one clustering with subtype A1 (n = 2) and CRF11 (n = 1). Finally, env sequences clustered with CRF02 (n = 10), and subtypes D (n = 2) and A1 (n = 1).
Out of the 18 strains for which at least two genomic fragments were available, 13 strains exhibited a similar subtype but clustering in different trees: 12 CRF_02, 1 D (Table 1). Three of the remaining sequences showed a discordant clustering pattern: two sequences were Apol/Denv, whereas one was F2gag/Apol/Aenv. Two strains showed only slight variations between different trees: strain Cam669 clustered within the CRF11 subcluster in gag and pol trees and within the CRF02 subcluster in env. In the V3–V5 region both CRF11 and CRF02 have a subtype A structure [11–14]; similarly, Cam532 clustered with CRF02 strains in pol and with subtype A1 in env. In this region CRF02 has an A1 structure [13,14]. Altogether, our results showed a relatively limited HIV-1 diversity in syringe washings from the southwest province of Cameroon. Few unique recombinant forms were detected in our study. The major form identified is CRF02, which was recently reported to be expanding in west-central Africa . This is not surprising, because CRF02 was shown to be the dominant viral form in the epidemics in west-central Africa .
Transmission through unsterilized and reused needles and syringes was claimed to be a significant cause of HIV infection in Africa . However, it was recently reported that ‘unsafe medical injections can be confidently excluded as a major source of HIV infection’ . Studies of HIV survival in syringes have been carried out in western countries , and clearly showed a risk of HIV transmission through unsafe injections. Therefore, it was important to carry out controlled studies under real-world conditions to prove or disprove the theory that injections were safe, especially in resource-poor settings. This is the first such study showing the presence of potentially infectious HIV in needles and syringes in a real-world situation. Previous attempts failed to amplify HIV in discarded syringes in Ethiopia . Our strategy of collecting syringe washes immediately after syringe use and of extracting DNA/RNA from relatively large amounts of material may account for our results compared with previous studies.
A large number of ‘clinics’ exist in sub-Saharan Africa and their policies for injections are not subjected to systematic control. Therefore, they represent a potential risk of nosocomial transmission of HIV. By showing that HIV-1 RNA was present in more than 30% of syringes used for intravenous injections, we provided proof of concept that injection practices could account for a significant proportion of new HIV infections. PCR amplification of viral nucleic acid demonstrated that a high viral level was present in the syringe wash, certainly enough to transmit HIV . Although some have argued that viral nucleic acid presence does not necessarily demonstrate viable viral presence, it does demonstrate that viable virus could be present, or at least, had been present. Therefore, our results show that caution must still be exercised and that the belief that HIV may not be transmitted by re-used needles is dangerous to public health initiatives.
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