Bartonella quintana in Body Lice and Head Lice from Homeless Persons, San Francisco, California, USA

Persons with lice may be at increased risk for infection with this bacterium.

T he human body louse and human head louse are generally recognized as 2 subspecies of Pediculus humanus (P. h. humanus and P. h. capitis, respectively) that have distinct ecologic preferences (1). However, a recent genetic analysis was not able to show any differences between these 2 subspecies (2). The human body louse is a small, parasitic insect that lives on the body and in the clothing or bedding of its human host. Body lice feed only on human blood. In the United States, body lice infestations usually are found only on persons who do not have access to clean changes of clothes or bathing facilities (e.g., the homeless population).
Head lice also feed only on human blood and are found on the head. Head lice infestations occur most often in school children and may also occur in the homeless population where they may be transferred by pillow cases, hats, and combs. Body and head lice are morphologically indistinguishable by the unaided human eye. Body lice are most reliably differentiated from head lice by their presence on clothing or on parts of the body other than the head or neck. These lice spend most of their time on the clothing of an infested person, visiting the body up to 5 times a day to feed. The eggs (called nits) of body lice are cemented to clothing fibers and seams or, occasionally, to body hairs (3,4).
In addition to causing discomfort and irritation, body lice can transmit disease-causing pathogens. Bartonella quintana is a bacterium transmitted through body lice feces that are scratched into the skin by the host. This organism can cause trench fever, endocarditis, bacillary angiomatosis, peliosis, and chronic bacteremia in infected humans (3). Since 1992, B. quintana has been recognized as a reemerging infection in homeless populations in the United States and Europe, as well as an opportunistic pathogen in patients with AIDS (5). Infection with B. quintana can cause prolonged disability in immunocompetent persons and can be life-threatening in immunodeficient patients.
Studies of homeless persons seeking medical care in clinics and hospitals in the United States and France have found that 2%-20% of persons tested had antibodies against B. quintana (6)(7)(8)(9). In Tokyo, Japan, 57% of homeless patients had immunoglobulin (Ig) G titers >128 for B. quintana (10). A study in Marseille, France, found that 14% (10/71) of homeless patients who came to a hospital emergency department had blood cultures positive for B. quintana (11). In 1990, physicians in the San Francisco, California, Bay area recognized the link between Barto-nella spp. infections and bacillary angiomatosis (12,13) and bacillary peliosis hepatis (14). A subsequent study by Koehler et al. documented the occurrence of bacillary angiomatosis in 49 patients seen over 8 years (15). All patients in this study were infected with either B. quintana or B. henselae (the agent of cat-scratch disease), most casepatients were immunocompromised (92% had HIV infection), and B. quintana infection was associated with homelessness and body lice infestation. In a subsequent study of HIV-positive patients with fever in San Francisco, Koehler and others found that 18% of 382 patients were positive for Bartonella spp. (16).
The human body louse is currently thought to play a role in the transmission of B. quintana among homeless persons, much as it did during the epidemics of trench fever that occurred during World Wars I and II (3). In the aforementioned study in Marseille, France, in 1999, body lice from 3 (20%) of 15 homeless patients were positive for B. quintana by PCR (11). In Tokyo, Sasaki et al. examined clothing from 12 homeless persons for body lice (17). These authors found that lice from 2 (16.7%) of 12 homeless persons were positive for B. quintana by PCR (17). Furthermore, evidence now indicates that head lice may be involved in the transmission cycle of B. quintana (18). Homeless populations in urban areas in northern California are vulnerable to body lice infestation and may be at risk for B. quintana infection. We studied whether body and head lice from homeless populations in a northern California city are carrying B. quintana or other pathogenic Bartonella spp.

Materials and Methods
In 2007 and 2008, staff from the Vector-borne Diseases Section of the California Department of Public Health (CDPH) participated in San Francisco's Project Homeless Connect (SFPHC). Under the auspices of SFPHC medical services, hair, body, and clothing of homeless persons were inspected for lice. Any lice found on the head with the presence of nits were considered to be head lice. Any lice on the body or clothing were considered to be body lice. Most infested persons self-referred directly to the CDPH booth at this event, with the exception of 1 physician referral. Lice were collected by using forceps, identified, sorted by subspecies, and placed in screw-top vials filled with 95% ethanol. Only a portion of the total lice infesting a person were collected for testing. The lice were shipped to the Centers for Disease Control and Prevention (Fort Collins, CO, USA) for detection and identification of Bartonella spp.
Lice were pooled by host and then subspecies. Samples from hosts with >20 lice were further tested individually to obtain an estimate of Bartonella spp. prevalence in the lice. We tested 36 pools of body lice, 7 pools of head lice, 108 individual body lice, and 4 individual head lice. Individual or pooled (2-20 lice/pool) samples were suspended in 250 µL of sterile phosphate-buffered saline and homogenized in an MM300 mixer (Retsch, Newtown, PA, USA) for 8 min. DNA was extracted from the homogenates by using a Mini Kit (QIAGEN, Valencia, CA, USA) and the Blood and Body Fluid Spin Protocol according to the manufacturer's protocol with a few minor changes. A PCR was performed in 20 µL of the mixtures containing 4-20 ng of the extracted DNA, 20 µL of 2× Ampdirect Plus, 0.5 U of Ex Taq Hot Start Version (Takara Bio, Otsu, Japan), and 1 pmol of each primer. Bartonella DNA was amplified by using gltA (citrate synthase gene) and ftsZ (cell division protein gene) primers as reported (19,20) in a thermalcycler (iCycler; BioRad, Hercules, CA, USA). A strain of B. washoensis was used as a positive control, and sterile deionized water was used as a negative control. Using gel electrophoresis on a 2% agarose gel, we examined the PCR products for 900-bp (ftsZ) and 380-400-bp (gltA) fragments.
The PCR amplicon of each gene was purified by using a QIA quick PCR purification kit (QIAGEN). Direct DNA sequencing of the purified PCR amplicons was conducted by using the BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, CA, USA) with the specific primers described above on a Model 3130X Genetic Analyzer (Applied Biosystems). Sequence data of each gene were aligned and compared with type strains of Bartonella spp. in GenBank by using MegAlign software (DNA Star Inc., Madison, WI, USA).

Results
In 2007 and 2008, 138 homeless persons had consultations at the CDPH booth at the SFPHC event. Of these persons, CDPH staff observed 33 persons with body lice infestations (23.9%) and 624 lice were collected (mean 18.9 lice/infested host). Head lice infestations were detected in 12 (8.7%) persons and 70 lice were collected (mean 5.8 lice/infested host). Six persons (4.3%) had body lice and head lice infestations.
Host sample SFB16 showed no amplification of Bartonella DNA in its original test but when an additional 3 pools of 20 lice and 11 individual lice were tested, 1 pool of 20 lice was positive. This host had a massive body louse infestation; 91 lice were collected from his clothing. Host sample SFB27 was also negative in its first test of a pool of 20 lice; 7 additional lice tested afterwards produced a single detection of B. quintana DNA in a body louse (14%).
Samples from 1 person who was co-infested with body lice and head lice were positive for Bartonella DNA by the gltA gene PCR (SFB17, 1 pool of 20 lice) in body lice, but not in the head lice pool (SFH7, n = 4). Samples from another co-infested person were negative for Bartonella DNA in 1 pool of 5 body lice (SFB10). Bartonella DNA was detected in a pool of 12 head lice (SFH3, MIR 8.3%) (Tables 1, 2).
Thirteen (86.7%) and 12 (80.0%) body lice from host SFB6 had Bartonella DNA amplification by gltA and ftsZ, respectively. Twenty-five (59.5%) and 23 (54.8%) of individual body lice from host SFB24 had Bartonella DNA amplification by gltA and ftsZ, respectively. Two samples from hosts SFB6 and SFB24 were sequenced and found to be identical with B. quintana type strain Fuller for both genes. Host sample SFB13 had Bartonella DNA amplification for both genes, and showed a sequence identical to B. quintana type strain Fuller for the gltA product and 99.9% homology to the same type strain for the ftsZ product. One of the individual head lice samples, SFH2, showed positive amplification of the ftsZ and gltA genes. Sequencing showed that this sample was B. quintana type strain Fuller for the gltA product.

Discussion
Our study has shown that homeless persons in the San Francisco Bay area have body and head lice that harbor B. quintana type strain Fuller. Prevalence of B. quintana in body lice from homeless persons (33.3%) in our study was slightly higher than the prevalence reported by Sasaki et al. in Tokyo, where body lice in 2 (16.7%) of 12 homeless persons were infected with B. quintana (17). Furthermore, similar prevalence of B. quintana infection in body lice has been reported from Russia (12.3%) (21) and Marseille, France (20%) (11).
Although Sasaki et al. detected B. quintana DNA in head lice by using molecular detection methods, their samples came from children in Nepal who also had body lice (18). However, there is no strong evidence that head lice are vectors of this organism between human hosts. Moreover, Fournier and others tested 143 head lice from schoolchildren from 8 countries and found no B. quintana (22) We have detected B. quintana in head lice from persons without a known concurrent body louse infestation. Further work is needed to examine how homeless persons acquire lice and which groups may be predisposed to louse infestation and B. quintana infection.