One question about some bacterial infections: does it pass from mother to baby?
Abstract
Bartonella vinsonii subsp. berkhoffii, Bartonella henselae, or DNA of both organisms was amplified and sequenced from blood, enrichment blood cultures, or autopsy tissues from four family members. Historical and microbiological results support perinatal transmission of Bartonella species in this family. It is of clinical relevance that Bartonella spp. may adversely influence human reproductive performance.
CASE REPORT
At the time of Bartonella testing, the father, mother, and son were 47, 46, and 10 years of age, respectively. Prior to meeting in 1989, both parents were reportedly healthy. The father and mother were born and raised on Long Island, where the father was a carpenter and the mother a graphic designer. As children and teenagers, the father had daily contact with dogs and the mother had daily contact with cats and dogs; however, both parents reported minimal exposure to other animals or arthropod vectors. At 16 years of age, the mother was severely bitten and scratched multiple times while bathing a feral, flea-infested cat. The couple was married in 1991 and their twins, conceived following in vitro fertilization, were born by cesarean section in 1998. The twin sister died nine days after birth, due to hypoplastic left heart syndrome. The family relocated to Florida in 2000, when their son was 15 months old. After their marriage, the couple did not maintain a pet cat and did not acquire a dog until the summer of 2009.
Waxing and waning symptoms, primarily consisting of fatigue, headaches, and urogenital pain and straining while voiding developed in both parents shortly after they were married. In 1991, the mother had a cervical biopsy due to vaginal warts. Between 1992 and 1995, the mother was diagnosed with irritable bowel syndrome, interstitial cystitis, and infertility. By 1999, both parents were experiencing persistent symptoms, including frequent headaches, memory loss and confusion, irritability, insomnia, bladder dysfunction, and balance problems. Between 2000 and 2009, all three family members reported intermittent joint pain, muscle pain, restlessness, shortness of breath, episodic tachycardia, rashes, rectal puritis, hypersensitivity to various foods, fatigue, episodes of extreme anxiety and irritability, cognitive impairment, and stabbing urethral pain when voiding, attributed to candidiasis. The husband, a nonsmoker, also had a chronic cough for years and occasional urinary tract infections.
Family members had sought care from primary care physicians and several specialists, including an internist, gastroenterologist, urologist, infectious disease physician, neurotoxin expert, and several naturopaths. Based upon blood mercury results, both parents had dental fillings removed in 2001. During the 4 years prior to Bartonella testing, the family had been treated with cholestyramine for suspected toxic mold exposure in their home in Florida. Neither this treatment, removal of the dental fillings, nor thorough remediation of the household environment while in Florida resulted in symptomatic improvement. The husband and wife had also been treated with doxycycline, azithromycin, and ciprofloxacin for Chlamydia pneumoniae and Mycoplasma pneumonia and with nitazoxanide for Blastocystis hominis. None of these treatments elicited symptomatic improvement. Beginning in 2000, both parents were repeatedly treated with long courses (up to 1 to 3 years) of fluconazole and nystatin for oral or vaginal candidiasis, with improvement in urogenital symptoms coinciding with each treatment. For financial and medical reasons, the family moved back to New York in 2009. There was no familial history of irritable bowel syndrome, interstitial cystitis, infertility, or congenital heart defects. Despite three additional attempts at in vitro fertilization, the mother did not become pregnant again.
The son had severe colic as a baby and subsequently experienced frequent night walking, night sweats, hyperactivity and irritability, dark circles under his eyes, frequent ear infections and eye blinking. The son was tested for mercury toxicity, Mycoplasma, Chlamydia, and intestinal parasites and all test results were negative. He had infrequent to no contact with animals or arthropod vectors. Despite his medical problems, the son attended school and participated in age-appropriate activities.
In January 2009, the primary author was contacted by the mother, requesting Bartonella testing as a component of an IRB-approved study (North Carolina State University Institutional Review Board, IRB number 164-08-05). Testing for the father, mother, and son was performed using a previously described diagnostic platform that incorporates enrichment culture of patient blood samples in Bartonella alphaproteobacterial growth medium (BAPGM) (6, 7, 12, 24). In addition to Bartonella spp., this nonselective medium will isolate other common and fastidious bacterial species (8). Previously described 16S-23S intergenic spacer (ITS) region and polymerase beta subunit (rpoB) gene PCR assays were used for the detection of Bartonella sp. DNA in this study (11, 22). Four to six independent 16S-23S ITS PCRs were performed for each patient sample set. Specifically, DNA was extracted directly from EDTA anticoagulated blood and from serum samples. Simultaneously, 2 ml of the aseptically obtained blood was inoculated into 10 ml of BAPGM as an enrichment culture step. Following incubation for 7 to 14 days in liquid culture medium, DNA was extracted for PCR testing. Subcultures on blood agar were incubated in 5% CO2 for up to 5 weeks. Archived paraffin-embedded postmortem tissues from the female twin and cervical and placental biopsy tissues from the mother were obtained for PCR testing and DNA sequencing. Each tissue was processed at three different time points, with extreme care taken to avoid DNA carryover or PCR amplicon contamination (29). Our laboratory took several precautionary approaches to minimize and to potentially eliminate the risk of PCR contamination and to prevent the carryover of either genomic or preamplified PCR products between samples. (i) Each component of sample processing (DNA extraction, PCR preparation, PCR amplification, and gel analysis) was performed in three physically separated rooms. A unidirectional work flow was enforced whereby any person handling materials or solutions in the PCR amplification and gel analysis room was not allowed to return to or perform work in the other two rooms. (ii) Positive controls consisting of 0.001 picograms per microliter of Bartonella genomic DNA (equivalent to 0.5 genome copies per microliter) were used for all PCR runs. This represents only twice the amount of template DNA that can be amplified by our protocols. A low-DNA-concentration control was purposely used in order to assess PCR performance and to minimize the potential for DNA carryover from the positive control. (iii) A negative control, consisting of Bartonella-free host DNA, was tested with every PCR run. To further ensure that any DNA template carryover from the positive-control sample would be detected, the negative control was prepared and allocated just after the positive-control sample and before any patient samples were processed in each PCR run. During the course of this study, a Houston 1-ITS strain of Bartonella henselae was exclusively used as the positive control. No contamination was detected in any of the 300 negative-control samples processed during the year in which the patient samples in this study were tested.
Serology was performed using modifications of a previously described indirect fluorescent antibody test (10). Bartonella vinsonii subsp. berkhoffii and B. henselae antibodies were determined following traditional immunofluorescence antibody assay (IFA) practices with fluorescein-conjugated goat anti-human IgG and B. vinsonii subsp. berkhoffii genotypes I, II, and III and B. henselae (Houston I strain) antigens. Sera were screened at dilutions of 1:16 to 1:256, and positives were further tested with 2-fold dilutions out to a final dilution of 1:8,192.
B. henselae (SA2 strain) DNA was amplified and sequenced from the mother's serum sample, and B. vinsonii subsp. berkhoffii genotype II DNA was amplified and sequenced from the enrichment blood cultures from the father (day 14 culture) and the mother (day 7 and 14 cultures) (Table 1). The son's initial BAPGM platform results were negative. Because azithromycin was administered for a sinus infection 1 week prior to obtaining blood for culture, the son was again tested for Bartonella bacteremia 8 and 20 weeks later (Table 1). ITS PCR results for the son's 8-week blood and serum samples were positive; however, direct sequencing generated partially overlapping sequences from each sample, with the nonoverlapping sequence consistent with B. henselae (SA2 strain). After ITS cloning, only B. henselae (GenBank accession number HMO42284) DNA was sequenced from the blood sample. However, using rpoB primers, followed by cloning, B. vinsonii subsp. berkhoffii (GenBank accession number accession number HMO42287) DNA was amplified and sequenced from the son's serum sample and from the enrichment blood culture (GenBank accession number HMO42288) (GenBank accession number HMO42284). Repeat testing, at 20 weeks, resulted in sequencing B. henselae DNA from the BAPGM enrichment culture. The father and mother were not seroreactive to any of the four Bartonella spp. test antigens, whereas the son was seroreactive to B. vinsonii subsp. berkhoffii genotypes I, II, and III and B. henselae antigens (Table 1). The family's pet dog, a 5-month-old male mixed breed acquired in 2009, was seronegative and PCR negative for all components of the BAPGM platform. All uninoculated BAPGM culture and PCR negative controls remained negative throughout the study.