Invisible Bleeding from Clean-Shave Haircuts: Detection with Blood Specific RNA MarkersKhumalo N.P.a · Mkentane K.a, b · Muthukarapan C.c · Hardie D.d, e · Korsman S.d, e · Hu N.d, f · Mthebe T.g · Davids L.M.b · Rousseau J.c
aDivision of Dermatology, Groote Schuur Hospital, Departments of bHuman Biology, cGenetics and dMedical Virology, Department of Clinical Laboratory Sciences, University of Cape Town, eNational Health Laboratory Service, Groote Schuur Hospital, fNational Institute for Communicable Diseases, University of Cape Town, and gLanga Community Health Clinic, Cape Town Central Health District, Western Cape, Cape Town, South Africa Corresponding Author
Background: ‘Haircut-associated bleeding' is a newly recognized entity that affects at least a quarter of African men who wear shiny clean-shave (‘chiskop') haircuts. Aim: This pilot study aimed to elucidate whether invisible haircut-associated bleeding was detectable using blood specific RNA markers (16 participants, 5 with unknown HIV status) and whether surface virus could be detected using PCR from scalp swabs (of 11 known HIV-positive participants). Methods: Haircuts were performed professionally and scalps examined by a dermatologist to exclude injury. Serum samples for viral loads were also collected at the same time. Results: In all, 6/16 (37%) samples tested positive (>100 relative fluorescent units) for hemoglobin beta and albumin, confirming evidence of blood; of these, only 1/11 was HIV-positive but had an undetectable serum viral load. No surface HIV was detected from any scalp samples. Conclusions: This study confirms the entity of haircut-associated bleeding but goes further to show for the first time that invisible bleeding from clean-shave haircuts is also common. Both a high serum viral load and evidence of bleeding should ideally be present prior to surface HIV detection. Future investigations for potential HIV (and hepatitis B) transmission through clean-shave haircuts are warranted but should not delay public education for disease prevention.
© 2013 S. Karger AG, Basel
Most haircuts are performed with scissors or clippers held a distance away from the scalp. Close-shave haircuts are worn by 70% of African men in the Cape Town ; the most popular of these is the shiny clean-shave cut, locally called ‘chiskop'. This is achieved by pressing the metal of the electric hair clipper directly and firmly onto the scalp without using the manufacturer-supplied clipper combs that determine hair length; the result is similar to that achieved with razor blades.
A history of visible bleeding after clean-shave haircuts has been reported in at least a quarter of men who wear this hairstyle (32% of men with unknown HIV status  and 24.8% of HIV-positive males ). Blood-borne viruses like the human immunodeficiency virus (HIV) are a major public health concern. Published evidence suggests that HIV remains viable for several days at room temperature in dried blood on surfaces  and may be resistant to commonly used disinfectants . The potential risk of virus transmission through non-sexual routes such as contaminated barbering equipment could easily be eliminated through adequate sterilization; there is however evidence of inconsistent disinfection and little or no sterilization of haircut equipment [6,7,8]. Many makeshift barbershops in Africa operate out of old shipping containers and shacks with no running water.
Several studies have demonstrated that the identification of body fluids such as invisible blood from forensic stains using mRNA blood specific markers is possible. These mRNA markers detect genes expressed in blood, i.e. hemoglobin beta (HBB), spectrin beta (SPTB) and porphobilinogen deaminase (PBGD) . The current pilot studies investigated whether evidence of invisible bleeding could be demonstrated from clean-shave haircuts, which RNA marker would be the most sensitive and whether HIV could be detected through PCR done on scalp swabs taken immediately after a haircut in HIV-positive participants.
Ethical approval was obtained from the Faculty of Health Science Ethics Committee and permission to conduct the study from the District Health Office. Informed consent was obtained from the study participants, who were all male and older than 18 years. The first pilot study investigated whether invisible bleeding or oozing of serum occurred. It included 5 participants of unknown HIV status who all had two samples taken, one tested for RNA markers (HBB, SPTB, PBGD) and a second for albumin. The second pilot study included 11 HIV-positive males who had a recent CD4 count (<500, preferably <200 if possible). Two scalp samples were taken from each, one for HBB and the other for HIV PCR, as well as whole blood for HIV PCR and CD4 count. Scalp samples were collected immediately after a clean-shave haircut performed by a professional barber. A dermatologist examined the scalp to ensure that no fluid (clear or red) was visible in included participants. Two sterile cotton swabs were rubbed over the subject's scalp and individually stored dry in a sterile tube for later use.
Total RNA was extracted as per manufacturer's instruction (Qiagen, South Africa). The eluted RNA was used for cDNA synthesis with reverse transcriptase (Fermentas, South Africa) and random hexamer primers. PCR was performed on blood lysates using sequence-specific primers for HBB, SPTB and PBGD (sequences available on request). PCR products were detected with a 3130xl Genetic Analyzer (Applied Biosystems, USA). Briefly, a 1-µl aliquot of PCR product was added to 8 µl Hi-Di Formamide and 0.2 µl of GeneScan 500 Rox size standard (Applied Biosystems). The samples were heated to 96°C before being loaded onto the analyzer. The conditions used for capillary electrophoresis were as follows: samples were injected through a 36-cm capillary filled with pop7 polymer at a temperature of 60°C. Samples were injected for 3 s using an injection voltage of 1.2 kV and electrophoresed for approximately 20 min. The raw data were analyzed using Peak Scan V1.0. Two sets of primers were added at the same time - one for the blood specific marker and a housekeeping gene (GAPDH) - to confirm whether a negative reaction really had no product or whether the reaction itself failed.
Albumin was detected using UV Spectrophotometry at 400-465 nm. Briefly, swabs were soaked in PBS for 4 h with intermittent vortexing before being centrifuged and then analyzed directly for microalbumin.
Viral loads for HIV RNA (quantitative real-time RNA PCR) were performed using the Abbott RealTime HIV-1 (Abbott Molecular Inc., USA) according to the manufacturer's protocol. In addition, a sensitive nested qualitative HIV RNA PCR amplified a 170 bp region of the gag gene and was performed using primers GAG A, GAG B, GAG C and GAG D as well as a PCR protocol based on Engelbrecht and van Rensburg . PCR products were analyzed by agarose gel electrophoresis.
Fluorescent capillary electrophoresis showed that the threshold for positive markers was 100 relative fluorescent units. The threshold value for a positive test was set to 100 fluorescent units based on a detection limit of 25 fluorescent units. At this level the background was minimal and peaks clear. In the first pilot study, the 5 samples tested were all positive for both HBB and albumin whilst other RNA markers were negative or inconsistently positive (table 1); samples were analyzed alongside a positive (blood) and a negative (blank swab) control (fig. 1). In the second pilot study, the HBB marker was positive in only 1 of 11 participants of whom the majority were on HIV treatment. Five of the 11 participants had a blood CD4 <200 and 9 had very low or undetectable blood HIV viral loads. In all samples, including the 2 participants with high blood viral loads, HIV was not detectable from scalp swabs (both qualitatively and quantitatively). Six out of 16 (37%) samples demonstrated the presence of HBB (table 2).
|Table 1. Pilot study 1: unknown HIV status|
|Table 2. Pilot study 2: HIV-positive participants|
|Fig. 1.a Positive control (known blood). Expected peak for HBB-positive samples at 152.37 bp using the HBB mRNA marker. b HIV-positive participant (scalp swab). Negative for blood specific mRNA marker (HBB), indicated by an absence of peak. c HIV-positive participant (scalp swab). Positive for blood specific mRNA marker (HBB), indicated by presence of peak at 152 bp. d Negative (RNA) control (clean swab). No blood identification as indicated by absence of RNA marker peak.|
Although no visible bleeding was observed in participants, blood was detected in 37% of samples. The 100% presence of HBB and albumin in the first 5 pilot study participants created the expectation of a high rate of HBB-positives for the second study. It was prematurely considered unnecessary to test for albumin as none of the samples in the first study were positive for albumin alone (which would suggest oozing of serum). All scalp swabs were taken (at the same time as the blood) without prior knowledge of current blood viral loads. We aimed to include treatment-naïve participants presenting with low CD4 counts (ideally <200) in order to estimate the worst possible risk of transmission potential. It is perhaps an indication of the success of the Langa HIV Clinic's diagnosis and treatment program that it was difficult during the study period to find new clinic attendees with CD4 values <200 (5 of 11 participants); only 2/5 were treatment-naïve and both tested HBB-negative. It was not surprising that all scalp swabs had undetectable virus, as even the one swab that was HHB-positive came from a participant with very low blood viral load.
We included the more sensitive nested qualitative test because commercial HIV viral load kits may not be reliable for virus detection in testing minute scalp samples. In hindsight, the study design was not suitable for detecting scalp surface HIV as a potential for disease transmission through haircut-associated injury. Ideally, swab HIV detection should be attempted in samples with proven haircut-associated bleeding and high serum viral loads. Future studies should also investigate the risk of hepatitis B transmission through contaminated clean-shave haircuts.
This study confirms data reporting that clean-shave haircuts increase the risk of injury and bleeding; it goes further to report for the first time that invisible bleeding also commonly occurs. Future studies to quantify the potential for HIV transmission through contaminated clean-shave haircuts should not delay urgent health education. The available data are adequate for pragmatic promotion of public health messages of adequate equipment sterilization between haircuts and promotion of individual clipper ownership. Further, chemical depilatories or longer haircuts are alternatives for avoiding haircut-associated injuries.
The authors are grateful to Sr. Petlho, Sr. Kwini, Mr. Joboda and the rest of the Langa Clinic staff, who made the study possible. They also thank Prof. Peter Berman for assistance with the determination of albumin in swab samples.
Lab consumables were funded through N.P.K., recipient of the Stiefel Award 2012 (South Africa).
The authors declare no conflict of interest.
Prof. Nonhlanhla P. Khumalo
Groote Schuur Hospital and The University of Cape Town
Observatory 7925 (South Africa)
Received: March 18, 2013
Accepted after revision: June 4, 2013
Published online: October 16, 2013
Number of Print Pages : 5
Number of Figures : 1, Number of Tables : 2, Number of References : 10
Vol. 227, No. 3, Year 2013 (Cover Date: November 2013)
Journal Editor: Saurat J.-H. (Geneva)
ISSN: 1018-8665 (Print), eISSN: 1421-9832 (Online)
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