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Parachlamydia and Rhabdochlamydia in premature neonates.

To the Editor: New members have recently been recognized in the order Chlamydiales (1). The family Rhabdochlamydiaceae includes R. porcellionis (a parasite of Porcellio scaber) and R. crassificans (a pathogen of the cockroach Blatta orientalis) (2,3); their pathogenic role in humans has not yet been investigated. Parachlamydia acanthamoebae and Protochlamydia naegleriophila belong to the family Parachlamydiaceae (1,4). Increasing evidence indicates that these obligate intracellular bacteria infecting free-living amebae may cause respiratory diseases in humans (1). Recent findings also suggest a role for Parachlamydia in miscarriage, stillbirth, and preterm labor (5-7). Whether these bacteria may contaminate the newborns of infected mothers is unknown.

The aims of this study were to 1) develop a real-time PCR for detecting Rhabdochlamydia spp. and 2) apply this PCR, and those previously described for Parachlamydia and Protochlamydia (4,8), to respiratory samples from premature neonates. Using the GenBank database (www.ncbi.nlm., we selected primers RcF (5'-GACGCTGCGTGAGTGATGA-3') and RcR (5'-CCGGTGCTTCTTT ACGCAGTA-3'), and probe RcS (5'-6 carboxyfluorescein-CTTTCGGGTTGTAAAACTCTTTCGCGCA-Black Hole Quencher 1-3'), which amplify parts of the 16S rRNA encoding gene, to specifically amplify Rhabdochlamydia spp. The 5'-FAM probe (Eurogentec, Seraing, Belgium) contained locked nucleic acids (underlined) to improve specificity. Reactions were performed with 0.2 [micro]M of each primer, 0.1 [micro]M of probe, and iTaq Supermix (Bio-Rad, Rheinach, Switzerland). PCR products were detected with ABI Prism 7000 (Applied Biosystems, Rotkreuz, Switzerland). Inhibition, negative PCR mixture, and extraction controls were systematically tested.

To enable quantification, a plasmid containing the target gene was constructed as described (4,9). The analytical sensitivity of the real-time PCR for Rhabdochlamydia spp. was [less than or equal to] 10 copies DNA/[micro]L. No cross-amplification was observed when the analytical specificity was tested with human, amebal (Acanthamoeba castellanii ATCC 30010), and bacterial DNA (online Technical Appendix, available from www.cdc. gov/EID/content/15/12/2072-Techapp. pdf). Intrarun and interrun reproducibility were excellent (online Technical Appendix).

This PCR and those previously described for Parachlamydia and Protochlamydia (4,8) were retrospectively applied to 39 respiratory samples from 29 neonates admitted in the neonatology unit of our institution (median 1 sample per patient, range 1-4 sample). All but 1 patient had a gestational age at birth [less than or equal to] 36 weeks (median 28.6, range 24.6-41.2 weeks). Respiratory distress syndrome was present in 25 (86%) of these 29 neonates. Samples had been drawn a median of 14 days (range 1-229 days) after birth, when clinically indicated. Results of PCR for Parachlamydia, Protochlamydia, and Rhabdochlamydia were positive for 9 (31%), 0 (0%), and 4 (14%) neonates, respectively. Positive results were obtained on the first sample drawn after birth for all but 2 neonates (initial negative results). One patient had positive PCR results for Parachlamydia and Rhabdochlamydia. These 12 newborns with positive PCR results for Parachlamydia and/or Rhabdochlamydia were compared with the 17 who had negative PCR results (Table).

Newborns with a Chlamydia-related organism documented in the respiratory tract had a significantly worse primary adaptation score (Apgar). These patients experienced more resuscitation maneuvers at birth. Durations of invasive mechanical ventilation and hospital stay were also longer among them. Three newborns died, compared with no deaths among the 17 with negative PCR results (p = 0.06). Pneumonia was documented in 5 of the 12 patients with positive Parachlamydia and/or Rhabdochlamydia PCR results but was concomitant to PCR positivity for only 3 of them. An alternative etiology was documented in all 3 (online Technical Appendix).

Parachlamydia and Rhabdochlamydia have thus been detected in a population of premature neonates. Most of these patients had severe respiratory distress syndrome, and the role of these bacteria as a causal agent of pneumonia could not be clearly assessed. The longer duration of mechanical ventilation for newborns with positive PCR results may suggest an occult superinfection with a Chlamydia-related bacterium contributing to the severity of the initial respiratory disease.

Our results also raise a question about the mode of acquisition of these microorganisms. A recent study reported a higher seroprevalence of Parachlamydia in women experiencing miscarriage (5,6), and DNA of this bacterium has been detected in the amniotic fluid of a woman with premature delivery (7). Whether neonatal infection results from a systemic infection during pregnancy or an inoculation at delivery is unknown. Because of the retrospective design of the study, no samples from the mothers were available for additional molecular or serologic analyses. Hospital water supplies are an important reservoir of free-living amebae and may represent another mode of acquisition because patients undergoing mechanical ventilation are exposed to aerosolized particles (10). Simultaneous detection of Parachlamydia and Rhabdochlamydia in 2 patients with initial negative results and their simultaneous detection in 1 neonate supports the latter hypothesis.

In conclusion, Parachlamydia and Rhabdochlamydia DNA were detected in respiratory secretions of premature newborns with more severe conditions at birth, more mechanical ventilation requirements, and a trend toward a higher mortality rate. The pathogenic role of these Chlamydia-related bacteria in neonates deserves further investigations.

This work was supported by the Swiss National Science Foundation grant FN 32003B-116445. G.G. is supported by the Leenards Foundation through a career award entitled Bourse Leenards pour la releve academique en medecine clinique a Lausanne. S.A. received the Analyses Medicales Services prize for the development of the Rhabdochlamydia PCR, under the supervision of G. Greub. This study was approved by the ethics committee of the University of Lausanne.

Frederic Lamoth, Sebastien Aeby, Antoine Schneider, Katia Jaton-Ogay, Bernard Vaudaux, and Gilbert Greub

Author affiliation: University Hospital and University of Lausanne, Lausanne, Switzerland

DOI: 10.3201/eid1512.090267


(1.) Greub G. Parachlamydia acanthamoebae, an emerging agent of pneumonia. Clin Microbiol Infect. 2009;15:18-28. DOI: 10.1111/j.1469-0691.2008.02633.x

(2.) Corsaro D, Thomas V, Goy G, Venditti D, Radek R, Greub G. Candidatus Rhabdochlamydia crassificans, an intracellular bacterial pathogen of the cockroach Blatta orientalis (Insecta: Blattodea). Syst Appl Microbiol. 2007;30:221-8. DOI: 10.1016/j.syapm.2006.06.001

(3.) Kostanjsek R, Strus J, Drobne D, Avgustin G. Candidatus Rhabdochlamydia porcellionis, an intracellular bacterium from the hepatopancreas of the terrestrial isopod Porcellio scaber (Crustacea: Isopoda). Int J Syst Evol Microbiol. 2004;54:543-9. DOI: 10.1099/ijs.0.02802-0

(4.) Casson N, Michel R, Muller KD, Aubert JD, Greub G. Protochlamydia naegleriophila as etiologic agent of pneumonia. Emerg Infect Dis. 2008;14:168-72. DOI: 10.3201/eid1401.070980

(5.) Baud D, Thomas V, Arafa A, Regan L, Greub G. Waddlia chondrophila, a potential agent of human fetal death. Emerg Infect Dis. 2007;13:1239-43.

(6.) Baud D, Regan L, Greub G. Emerging role of Chlamydia and Chlamydia-like organisms in adverse pregnancy outcomes. Curr Opin Infect Dis. 2008;21:70-6. DOI: 10.1097/QCO.0b013e3282f3e6a5

(7.) Baud D, Goy G, Gerber S, Vial Y, Hohlfeld P, Greub G. Evidence of maternal-fetal transmission of Parachlamydia acanthamoebae. Emerg Infect Dis. 2009;15:120-1. DOI: 10.3201/ eid1501.080911

(8.) Casson N, Posfay-Barbe KM, Gervaix A, Greub G. New diagnostic real-time PCR for specific detection of Parachlamydia acanthamoebae DNA in clinical samples. J Clin Microbiol. 2008;46:1491-3. DOI: 10.1128/JCM.02302-07

(9.) Jaton K, Bille J, Greub G. A novel real-time PCR to detect Chlamydia trachomatis in first-void urine or genital swabs. J Med Microbiol. 2006;55:1667-74. DOI: 10.1099/jmm.0.46675-0

(10.) Thomas V, Herrera-Rimann K, Blanc DS, Greub G. Biodiversity of amoebae and amoeba-resisting bacteria in a hospital water network. Appl Environ Microbiol. 2006;72:2428-38. DOI: 10.1128/ AEM.72.4.2428-2438.2006

Address for correspondence: Gilbert Greub, Center for Research on Intracellular Bacteria, Institute of Microbiology, Centre Hospitalier Universitaire Vaudois, Rue du Bugnon 46, CH1011 Lausanne, Switzerland; email: gilbert.
Table. Characteristics of 29 newborns with positive PCR results for
Parachlamydia acanthamoebae or Rhabdochlamydia spp. and controls *

                                            Positive PCR
Characteristics                            result, n = 12

Sex, M/F                                   8 (67)/4 (33)
Gestational age at birth, wk, median
  (range)                                    27 (24-36)
Weight <10th percentile                        4 (33)
Height <10th percentile                        3 (25)
Primary adaptation
  First Apgar score (1 min), median
    (range)                                  2.5 (0-7)
  First 3 Apgar scores, ([double
    dagger]) median (range)                 18.5 (8-27)
  Cardiac massage in first 48 h                6 (50)
  Endotracheal intubation in first 48 h       11 (92)
Respiratory distress syndrome                 11 (92)
Hyaline membranes disease                      9 (75)
Bradypneic syndrome                            7 (58)
Bronchopulmonary dysplasia                     8 (67)
Amniotic fluid aspiration                      1 (8)
Invasive mechanical ventilation, d,
  median (range)                             12 (2-50)
Endotracheal intubation during hospital
  stay                                        12 (100)
Infectious complications
  Lung infection                               5 (42)
  Other systemic infection                     7 (58)
Other complications
  Intraventricular hemorrhage                  4 (33)
  Persistent artery canal                      7 (58)
  Necrotizing enterocolitis                    2 (17)
  Congenital malformations                     1 (8)
  Stay in neonatology ward, d, [section]
    median (range)                         113.5 (9-435)
  Death                                        3 (25)
  Premature membranes rupture                  5 (42)
  Placental detachment                         2 (17)
  Preeclampsia                                 4 (33)
  Systemic infection                           7 (58)
  Cesarean delivery                            9 (75)

                                            Negative PCR     p value
Characteristics                            result, n = 17   ([dagger])

Sex, M/F                                   6 (35)/11 (65)      0.14
Gestational age at birth, wk, median
  (range)                                    30 (25-41)        0.16
Weight <10th percentile                        4 (24)          0.68
Height <10th percentile                        6 (35)          0.69
Primary adaptation
  First Apgar score (1 min), median
    (range)                                   8 (2-9)         0.0017
  First 3 Apgar scores, ([double
    dagger]) median (range)                  27 (17-29)       0.0023
  Cardiac massage in first 48 h                0 (0)          0.002
  Endotracheal intubation in first 48 h        8 (47)         0.019
Respiratory distress syndrome                 14 (82)          0.62
Hyaline membranes disease                      8 (47)          0.25
Bradypneic syndrome                           11 (65)          1.00
Bronchopulmonary dysplasia                     9 (53)          0.70
Amniotic fluid aspiration                      3 (18)          0.62
Invasive mechanical ventilation, d,
  median (range)                              3 (0-14)        0.005
Endotracheal intubation during hospital
  stay                                        11 (65)         0.028
Infectious complications
  Lung infection                               7 (41)          1.00
  Other systemic infection                     5 (29)          0.15
Other complications
  Intraventricular hemorrhage                  6 (35)          1.00
  Persistent artery canal                      6 (35)          0.27
  Necrotizing enterocolitis                    1 (6)           0.55
  Congenital malformations                     1 (6)           1.00
  Stay in neonatology ward, d, [section]
    median (range)                           48 (7-131)       0.003
  Death                                        0 (0)           0.06
  Premature membranes rupture                  6 (35)          1.00
  Placental detachment                         1 (6)           0.55
  Preeclampsia                                 2 (12)          0.20
  Systemic infection                           9 (53)          1.00
  Cesarean delivery                           10 (59)          0.45

* Results are given as no. (%) except as indicated.

([dagger]) Fisher exact test and nonparametric Mann-Whitney rank sum
test were used for the analysis of proportions and continuous
variables, respectively.

([double dagger]) Sum of the 3 scores at 1, 5, and 10 min after birth.

([section]) If survived.
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Article Details
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Title Annotation:LETTERS
Author:Lamoth, Frederic; Aeby, Sebastien; Schneider, Antoine; Jaton-Ogay, Katia; Vaudaux, Bernard; Greub, G
Publication:Emerging Infectious Diseases
Article Type:Report
Geographic Code:4EXSI
Date:Dec 1, 2009
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