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![SHOULD HIGHER VANCOMYCIN TROUGH LEVELS BE
TARGETED FOR INVASIVE COMMUNITY-ACQUIRED
METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS
INFECTIONS IN CHILDREN?
Natalia Jimenez-Truque, MQC, MSCI, Isaac Thomsen, MD,
Elizabeth Saye, BS, and C. Buddy Creech, MD, MPH
Abstract: Methicillin-resistant Staphylococcus aureus isolates with van-
comycin minimal inhibitory concentrations (MICs) Ն1.5 g/mL have been
associated with poorer clinical outcomes and treatment failures in adults.
We evaluated vancomycin MICs in 71 invasive pediatric community-
acquired MRSA isolates from 2004 to 2008, using the E-test micromethod
and the E-test macro-method. The modal MIC by micromethod was 1.5
g/mL, and median vancomycin MICs did not increase over time.
Key Words: MRSA, vancomycin, methicillin resistance, Staphylococcus
aureus, pediatric, invasive
Accepted for publication October 8, 2009.
From the Department of Pediatric Infectious Diseases, Vanderbilt Univer-
sity Medical Center, Nashville, TN.
Supported, in part, by Vanderbilt CTSA grant 1 UL1 RR024975 from
NCRR/NIH and Fogarty International Center grant 1 R25 TW007697.
Address for correspondence: C. Buddy Creech, MD, MPH, 1161 21st Ave
South, CCC-5311 Medical Center North, Nashville, TN 37232. E-mail:
buddy.creech@vanderbilt.edu.
DOI: 10.1097/INF.0b013e3181c52a04
Since the emergence of methicillin-resistant S. aureus (MRSA),
vancomycin has been a key antimicrobial agent for the treat-
ment of MRSA infections.1,2
Concerns surrounding the long-term
use of vancomycin as a primary therapy were confirmed when the
first vancomycin–intermediate S. aureus (VISA) isolate was diag-
nosed in Japan in 1996.3
Recently, it was shown in adult patients
that strains with minimal inhibitory concentrations (MICs) Ն1.5
g/mL, though not above the susceptibility breakpoint of 2 g/
mL, were associated with clinical failure.4,5
This increase in
vancomycin MICs over time is defined as MIC creep.6 – 8
Because of MIC creep, tissue penetration of vancomycin,
and other factors, the vancomycin MIC breakpoints were lowered
in 2006. According to these breakpoints, an isolate with an MIC of
Յ2 g/mL is considered susceptible to vancomycin, an isolate
with intermediate resistance has an MIC of 4 to 8 g/mL, and an
isolate with an MIC Ն16 g/mL is resistant. Some bacterial
colonies within the staphylococcal population, on exposure to
vancomycin, develop an intermediately resistant phenotype known
as heterogeneous-vancomycin intermediate S. aureus (hVISA), a
phenotype that may be responsible for treatment failures despite
overall vancomycin susceptibility.2,6
Most data regarding MIC creep has been collected from
adult isolates. It is unclear whether this phenomenon occurs in
MRSA isolates from pediatric patients, specifically those classified
as community-associated MRSA (CA-MRSA). CA-MRSA iso-
lates would be expected to have lower vancomycin MICs when
compared with hospital-associated MRSA isolates due to the lack
of selective vancomycin pressure in the community. We hypoth-
esized that vancomycin MICs have not changed significantly over
time in the pediatric population, because risk factors such as
frequent vancomycin exposure and foreign bodies such as cathe-
ters or prosthetic joints are not likely to be present in children with
CA-MRSA disease. To evaluate this, we studied the vancomycin
MICs for pediatric CA-MRSA isolates from 2004 to 2008 based
on site of infection.
MATERIALS AND METHODS
S. aureus Clinical Isolates. Since 2004, all pediatric CA-MRSA
isolates at Vanderbilt Children’s Hospital have been archived.
Each isolate represents a unique pediatric patient. Isolates are
considered to be CA-MRSA based on application of Centers for
Disease Control-criteria.9
For this study, we analyzed vancomycin
MICs for 71 previously characterized invasive pediatric CA-
MRSA isolates collected from 2004 to 2008 that were viable in
culture and in which site of infection and date were known. These
71 invasive CA-MRSA isolates were randomly selected from a
de-identified pediatric clinical isolate database of 1376 unique
isolates, using a random number generator. In 706 isolates, unam-
biguous notation of site of infection was available; from these, 80
were from patients with invasive MRSA disease, and 71 were
viable in culture and had molecular features characteristic of
CA-MRSA.
Isolates were initially classified as MRSA by the clinical
laboratory of Vanderbilt Children’s Hospital and subsequently
confirmed by our laboratory based on growth on mannitol salt agar
plates containing oxacillin and a positive latex agglutination test
for clumping factor (Staphaurex, Remel). DNA was extracted and
purified and was used as template for polymerase chain reaction
(PCR) detection of nuc and mecA genes and for staphylococcal
cassette chromosome mec (SCCmec) typing, as described else-
where.10
Genotyping of isolates was performed by pulse-field gel
electrophoresis and/or repetitive element sequence based PCR.11
E-Test Micro- and Macro-Methods. S. aureus strain ATCC 29213
was used as the reference strain for both E-test methods (AB-
Biodisk, Solna, Sweden), which were performed according to the
manufacturer’s guidelines. For the micromethod, a 0.5 McFarland
standard was prepared in sterile saline, inoculated onto Mueller-
Hinton agar, and incubated for 24 hours at 35°C. For the macro-
method, a 2 McFarland standard was inoculated onto brain-heart
infusion agar and incubated for 48 hours at 35°C. Testing of the
clinical isolates was done in a single laboratory, and the results
were recorded by a single observer. S. aureus isolates with van-
comycin MICs by micromethod of Յ2 g/mL were considered
susceptible (VSSA) based on Clinical and Laboratory Standards
Institute guidelines. Intermediate susceptibility to vancomycin
(VISA) was defined by MICs of 4 to 8 g/mL, and vancomycin
resistance (VRSA) by MICs of Ն16 g/mL.
Analysis of MICs over time and by site of infection was
performed using the Kruskal-Wallis H method. A P value of 0.05
was considered statistically significant. All analyses were per-
formed with SPSS Version 16.0.
RESULTS
To confirm that each of the 71 isolates clinically determined
to be community-associated were also genotypically consistent
with CA-MRSA, we performed SCCmec typing and genotyping by
pulsed-field gel electrophoresis or rep-PCR. All of the isolates had
SCCmec IV cassette and belonged to USA300, the current epi-
demic clone in the United States. Of the 71 invasive isolates, 47
(66.2%) were from joint infections (Table 1).
Overall, the modal MIC by micromethod was 1.5 g/mL.
Fifty-six isolates had an MIC Ն1.5 g/mL. Three isolates had
an MIC of 2 g/mL, but are considered susceptible by Clinical
and Laboratory Standards Institute breakpoints. Only 15 iso-
lates had an MIC Ͻ1.5 g/mL. Median and mean vancomycin
MICs did not increase over time (P ϭ 0.245, Inter-Quartile
Range [IQR] 1.5–1.5 g/mL). Similarly, MIC values were not
significantly different across different infection sites (P ϭ
0.952, IQR ϭ 1.5–1.5 g/mL).
Jimenez-Truque et al The Pediatric Infectious Disease Journal • Volume 29, Number 4, April 2010
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- 1. BRIEF REPORTS HUMAN RHINOVIRUSCAUSES SEVERE INFECTION IN PRETERM INFANTS Rene´e O. van Piggelen, MD,* Anton M. van Loon, PhD,† Tanette G. Krediet, MD, PhD,* and Malgorzata A. Verboon-Maciolek, MD, PhD* Abstract: Data of 11 infants (median gestational age and birth weight 30 weeks and 1520 g, respectively) with severe human rhinovirus infection (HRV) are described. Nine of 11 (82%) were preterm infants and 7 of these 9 (78%) became infected during their stay in the neonatal intensive care unit. All infants presented with respiratory distress and all needed respi- ratory support for a median of 6 days. Radiologic findings included perihilar streakiness, atelectasis, focal consolidation, and hyperinflation. The diagnosis of HRV infection was made by real-time polymerase chain reaction in nasopharyngeal aspirate. All infants recovered from their HRV infection. HRV can cause severe disease in preterm infants requiring respiratory support. Key Words: human rhinovirus, neonatal infection, preterm infant Accepted for publication October 20, 2009. From the Departments of *Neonatology, and †Virology, University Med- ical Center, Utrecht, The Netherlands. Address for correspondence: Malgorzata A. Verboon-Maciolek, MD, PhD, Department of Neonatology, University Medical Center, Lundlaan 6, 3584 EA Utrecht, KE 04.123.1, The Netherlands. E-mail: m.verboon- maciolek@umcutrecht.nl. Copyright © 2010 by Lippincott Williams & Wilkins DOI: 10.1097/INF.0b013e3181c6e60f Human rhinoviruses (HRV) are a major cause of common cold in adults and children.1 Usually HRV infections run a mild course, but it has recently been shown that HRV can be associated with severe lower respiratory tract infection in children.2 There are no published data available on HRV infections in preterm infants. We retrospectively analyzed the data of all neonates with con- firmed HRV infection admitted to our neonatal intensive care unit (NICU) from 2003 until 2008. MATERIALS AND METHODS We included all infants with a confirmed diagnosis of HRV infection and admitted from 2003, when the HRV real-time poly- merase chain reaction (PCR) was introduced in our hospital, until 2008. We analyzed the clinical, virologic, radiologic and labora- tory data of these infants. The diagnosis of HRV infection was made on the basis of a positive real-time PCR for HRV in a nasopharyngeal aspirate. Samples of all infants were also tested by real-time PCR for the following viral and bacterial pathogens: respiratory syncytial virus (RSV), human metapneumovirus, para- influenzaviruses 1 to 4, influenzaviruses A and B, human corona- viruses (HCoV: 229E, OC43, and NL-63), adenoviruses, Myco- plasma pneumoniae, Chlamydia pneumoniae, and since 2006 bocavirus. All PCRs were real-time assays based on TaqMan probes. The target for the HRV PCR was the 5Ј noncoding region of the genome. The amount of virus in each sample was recorded semi-quantitatively based on the cycle threshold (Ct) value of the sample in the PCR. The Ct value indicates the number of cycles needed in PCR before a sample becomes positive, and is therefore directly related to the amount of viral genome in the sample. Thus, a low Ct value corresponds to a high viral load, and a high Ct value to a low viral load. HRV was recorded as the dominating virus in a sample when the Ct value was at least 4 cycles lower than the Ct value for any other virus. Ct values above 45 were considered to be negative. Molecular typing of HRV was not performed. RESULTS During the study period viral infection was suspected in 62 infants admitted to our NICU. Depending on the clinical presen- tation (sepsis, meningitis, or pneumonia) we looked for viral agents of infection in different clinical samples. In patients with pneumonia we performed a PCR on respiratory viruses in naso- pharyngeal aspirate as described in the methods section. In 8 of 62 infants, we could not identify any virus. In 22 (41%) of 54 infants with a proven viral infection, we identified 24 respiratory viruses. The most frequently detected viruses were HRV (n ϭ 11) and RSV (n ϭ 8). The remaining respiratory viruses were HCoV (n ϭ 2), influenza virus (n ϭ 1), adenovirus (n ϭ 1), and parainfluenza virus (n ϭ 1). In 1 infant, 3 different respiratory viruses (HRV, RSV, and HCoV) were found. The characteristics of 11 infants with HRV infection are shown in Table 1. Nine of 11 infants (82%) were born prematurely with a median gestational age of 30 weeks (range: 26–32 weeks). The other 2 infants were born at term (39 and 41 weeks, respec- tively). One underwent surgical correction because of a diaphrag- matic hernia, whereas the other was admitted from home at 5 days of age. The median age at onset of symptoms was 49 days (range: 5–94 days). In 7 of 11 infants HRV infection was acquired during their hospital stay. The main presenting symptoms were respiratory distress (9/11), apnea (7/11), rhinorrhea (6/11), and hypothermia (5/11). All infants required respiratory support for a median of 6 days (range: 3–11 days), in 9 infants mechanical ventilation was nec- essary. The C-reactive protein was slightly elevated at onset of symptoms with a median value of 15 mg/L, and the white blood cell count was consistently normal. The Ct value of HRV was low in all infants with a median of 21 (range: 18.4–28.8) at onset of symptoms, indicating the presence of high viral loads of HRV in the respiratory tract. In 2 infants (patient 9 and 11), the Ct value increased from 24.6 to 29.4 and from 18.4 to 35.6, respectively, in samples taken 7 days later. Chest radiographs revealed perihilar streakiness (10/11), atelectasis (9/11), focal consolidation (6/11), and hyperinflation (6/11). Most infants had 2 or more of the above mentioned radiologic findings. One patient (patient 4) had a coinfection with 2 other viral pathogens, HCoV and RSV, but the viral loads of RSV (Ct: 35.1) and HCoV (Ct: 31.2) were consid- erably lower than that of HRV (Ct: 26.5). We did not find any nosocomial spread of HRV among our infants. Our patients were not clustered in time, but represented separate events. All infants recovered from the episode of HRV infection, but 4 patients (1 term and 3 preterm) subsequently developed recurrent lower re- spiratory tract infections. DISCUSSION Our study shows that HRV can cause severe pulmonary disease among infants admitted to a NICU. HRV is generally known as the causative agent of the common cold. The association of HRV with asthma exacerbations, wheezing, and lower respira- tory tract infections has been well recognized.2– 4 Recently, HRV also appeared to be an important reason for hospitalization in young children.5–7 However, HRV can also be detected in asymp- tomatic children.8 It is suggested that the identification of HRV in asymptomatic infants represents low-level infection without clin- ical symptoms, or is a first sign of developing illness.5 Data on HRV infections in very young infants are limited. In our previous retrospective analysis (1992–2003) of viral infections in our NICU, we found that HRV contributed to 2% of all proven viral infections among admitted infants.9 This percentage recently in- creased to 20% (unpublished data), which can be explained by the The Pediatric Infectious Disease Journal • Volume 29, Number 4, April 2010364 | www.pidj.com
- 2. introduction of real-timePCR for the identification of viruses in infants with severe respiratory disease. The clinical presentation of HRV infection in infants de- scribed in our study did not differ from that of RSV infection.10,11 All infants developed rhinorrhea, respiratory distress, apnea, and hypothermia, and required respiratory support for 3 to 11 days. It was not possible to differentiate between RSV and HRV infection based on radiologic findings. The presence of atelectasis which changed position daily and the huge production of sputum were common findings. We recorded relatively low Ct values corre- sponding to relatively high viral loads in the respiratory samples of our patients. In one infant (patient 4), who was infected with 3 different respiratory viruses, HSV was considered the major caus- ative agent because of its lowest Ct value. In 2 infants the Ct value performed again after 7 days was increased significantly, which was associated with clinical recovery. All infants recovered, but 4 developed recurrent respiratory tract infections which could not be explained solely by prematurity. In conclusion, HRV can cause severe pulmonary disease in preterm infants, requiring respiratory support. REFERENCES 1. Brownlee JW, Turner RB. New developments in the epidemiology and clinical spectrum of rhinovirus infections. Curr Opin Pediatr. 2008;20: 67–71. 2. Louie JK, Roy-Burman A, Guardia-Labar L, et al. Rhinovirus associated with severe lower respiratory tract infections in children. Pediatr Infect Dis J. 2009;28:337–339. 3. Tan WC. Viruses in asthma exacerbations. Curr Opin Pulm Med. 2005;11: 21–26. 4. Kusel MM, de Klerk NH, Holt PG, et al. Role of respiratory viruses in acute upper and lower respiratory tract illness in the first year of life: a birth cohort study. Pediatr Infect Dis J. 2006;25:680–686. 5. Jartti T, Lee WM, Pappas T, et al. Serial viral infections in infants with recurrent respiratory illnesses. Eur Respir J. 2008;32:314–320. 6. Miller EK, Lu X, Erdman DD, et al. Rhinovirus-associated hospitalizations in young children. J Infect Dis. 2007;195:773–781. 7. Piotrowska Z, Vazquez M, Shapiro ED, et al. Rhinoviruses are a major cause of wheezing and hospitalization in children less than 2 years of age. Pediatr Infect Dis J. 2009;28:25–29. 8. Wright PF, Deatly AM, Karron RA, et al. Comparison of results of detection of rhinovirus by PCR and viral culture in human nasal wash specimens from subjects with and without clinical symptoms of respiratory illness. J Clin Microbiol. 2007;45:2126–2129. 9. Verboon-Maciolek MA, Krediet TG, Gerards LJ, et al. Clinical and epide- miologic characteristics of viral infections in a neonatal intensive care unit during a 12-year period. Pediatr Infect Dis J. 2005;24:901–904. 10. Prodhan P, Westra SJ, Lin J, et al. Chest radiological patterns predict the duration of mechanical ventilation in children with RSV infection. Pediatr Radiol. 2009;39:117–123. 11. Forster J, Schumacher RF. The clinical picture presented by premature neonates infected with respiratory syncytial virus. Eur J Pediatr. 1995;154: 901–905. TABLE 1. Clinical, Laboratory, Virologic and Radiologic Data of 11 Infants With HRV Infection Patient/Birth/Sex GA (wk) BW (g) Onset (d) Clinical Signs CRP at Onset (max) (mg/L) WBC Count at Onset ϫ109 /L Ct Value Respiratory Support (d) Chest Radiograph 1/October 2003/M 29 1495 34 Apneas, rhinorrhea, respiratory distress 9 (48) 7.0 28.3 4 Atelectasis, perihilar streakiness focal consolidation 2/February 2005/F 32 1900 18 Respiratory distress 17 (37) 16.7 23.4 6 Atelectasis, perihilar streakiness, hyperinflation 3/November 2005/M 30 1520 75 Hypothermia, rhinorrhea, respiratory distress 79 (81) 8.9 22.9 6 Atelectasis, perihilar streakiness, hyperinflation, focal consolidation 4/November 2005/F 32 2095 28 Hypothermia, apneas, respiratory distress 15 (109) 16.7 26.5 6 Atelectasis, perihilar streakiness, hyperinflation 5/May 2006/M* 32 1700 49 Respiratory distress 17 (17) 18 23.9 11 Atelectasis, perihilar streakiness, hyperinflation, focal consolidation 6/April 2006/F 28 895 90 Hypothermia, apneas 27 (27) 4.5 28.8 5 Atelectasis, perihilar streakiness 7/October 2006/M 29 970 94 Apneas, rhinorrhea, respiratory distress 2 (6) 9.5 27.1 7 Atelectasis, perihilar streakiness, hyperinflation, focal consolidation 8/November 2006/M 41 3050 5 Hypothermia, respiratory distress 16 (21) 10.6 19.5 3 Atelectasis, perihilar streakiness 9/April 2007/F 30 1150 45 Hypothermia, apneas, rhinorrhea 6 (6) 11.0 24.6 7 Perihilar streakiness 10/September 2007/M† 39 3480 50 Apneas, rhinorrhea, respiratory distress 2 (2) 7.7 20.5 11 Atelectasis, hyperinflation focal consolidation 11/December 2007/F‡ 26 890 68 Apneas, rhinorrhea, respiratory distress 2 (2) 10.1 18.4 8 Perihilar streakiness, focal consolidation *Patient 5: VACTERL anomaly. † Patient 10: congenital diaphragmatic hernia. ‡ Patient 11: chronic lung disease. The Pediatric Infectious Disease Journal • Volume 29, Number 4, April 2010 Human Rhinovirus Infection in Preterm Infants © 2010 Lippincott Williams & Wilkins www.pidj.com | 365
- 3. CLINICAL PERFORMANCE OFA RAPID INFLUENZA TEST AND COMPARISON OF NASAL VERSUS THROAT SWABS TO DETECT 2009 PANDEMIC INFLUENZA A (H1N1) INFECTION IN THAI CHILDREN Piyarat Suntarattiwong, MD,* Richard G. Jarman, PhD,† Jens Levy, PhD,‡ Henry C. Baggett, MD, MPH,§ Robert V. Gibbons, MD, MPH,† Tawee Chotpitayasunondh, MD, DTM&H,* and James M. Simmerman, PhD, RN‡ Abstract: We identified febrile pediatric outpatients seeking care for influenza like illness in Bangkok. Two nasal and 1 throat swab were tested using the QuickVue AϩB rapid influenza kit and reverse transcription-polymerase chain reaction. Among 142 pandemic influenza A (H1N1)-positive patients, the QuickVue test identified 89 positive tests for a sensitivity of 62.7% (95% confidence interval ͓CI͔: 54.7–70.6). Specificity was 99.2% (95% CI: 98– 100). In the 0 to 2 years age group, sensitivity was 76.7% (95% CI: 61.5–91.8). Throat and nasal swabs are equally useful diagnostic specimens for reverse transcription-polymerase chain reaction diagnosis. Key Words: influenza, rapid diagnostic tests, pandemic, Thailand Accepted for publication October 20, 2009. From the *Queen Sirikit National Institute of Child Health Department of Medical Service, Ministry of Public Health, Bangkok, Thailand; †US Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand; ‡Influenza Division, International Emerging Infections Program, Thailand MOPH-US CDC Collaboration, Nonthaburi, Thailand; and §International Emerging Infections Program, Thailand MOPH-US CDC Collaboration, Nonthaburi, Thailand. The opinions or assertions contained herein are the private views of the authors and are not to be construed as official, or as reflecting the views of the US Centers for Disease Control and Prevention, the Department of the Army or the Department of Defense. Address for correspondence: James M. Simmerman, PhD, RN, International Emerging Infections Program, Thailand MOPH-US CDC Collaboration, Box 68 CDC, APO AP 96546, Nonthaburi, Thailand. E-mail: msimmerman@cdc.gov. DOI: 10.1097/INF.0b013e3181c6f05c Anovel quadruple reassortant influenza A/H1N1 virus emerged in North America in early 2009 and rapidly spread to hundreds of countries around the world causing the World Health Organi- zation to declare a pandemic on June 11.1 Following sporadic infections in returning Thai travelers in May and June, community transmission was established and 14,976 cases and 119 deaths due to laboratory-confirmed 2009 pandemic influenza A (H1N1) virus infection were reported in Thailand by August 22, 2009.2 Rapid laboratory diagnosis of influenza can improve clinical care, facil- itate outbreak investigations, and support infection control mea- sures. Highly sensitive real-time reverse transcription-polymerase chain reaction (RTPCR) assays are the preferred diagnostic method but are complex and costly, often not available in devel- oping countries, and results are seldom available in time to influence treatment decisions. In contrast, commercially available rapid influenza diagnostic tests (RIDTs) are simple, produce re- sults in a few minutes, and are widely used to detect influenza infections in outpatient settings. Broadly, RIDTs have been found to have high specificity (76%–100%) but low to moderate sensi- tivity (10%–100%) to detect seasonal influenza viruses.3 We investigated the sensitivity and specificity of the Quick- Vue AϩB rapid influenza test to detect 2009 pandemic influenza A (H1N1) virus infection compared with real time RTPCR in febrile pediatric outpatients in Bangkok, Thailand. We also studied whether nasal or throat swabs produce superior specimens to detect the 2009 pandemic virus by RTPCR. MATERIALS AND METHODS The study was approved by the Ethical Review Committee of the Queen Sirikit National Institute of Child Health. During July 2009 and August 2009, we prospectively identified and obtained written consent from the guardians of pediatric patients who sought outpatient care for influenza-like illness (ILI). For children less than 2 years of age, ILI was defined as fever Ն38°C and one or more of the following symptoms: nasal discharge/congestion, cough, conjunctivitis, respiratory distress, sore throat, new seizure. For children Ն2 years of age ILI was defined as fever Ն38°C and cough or sore throat. Eligible patients were 1 month to 15 years of age. Rapid test results were immediately provided to the attending physician to support treatment decisions. RTPCR was used as the standard to calculate the performance of the rapid test. We ex- cluded subjects who tested positive by RTPCR for seasonal influ- enza strains from the analysis. We derived 95% confidence limits for sensitivity and specificity using the Wald-normal-approxima- tion using R version 2.8.1. We used the simple kappa coefficient to test for agreement between throat and nasal swabs by RTPCR. Specimens (2 nasal and 1 throat) were collected from each child by hospital nurses experienced in collecting respiratory swab specimens. The foam-tipped nasal swab provided by the manufac- turer was immediately tested according to manufacturer instruc- tions using the QuickVue Influenza AϩB rapid diagnostic kit (Quidel Co., San Diego, CA). The remaining Dacron-tipped nasal and throat swabs were immediately placed in separate M4RT viral transport media (Remel, Lenexa, KS), and sent the same day on wet ice to the Armed Forces Institute of Medical Sciences. These specimens were aliquoted within 24 hours and then stored at Ϫ70°C until processed for RTPCR. Viral ribonucleic acid was extracted from 140 L of inoculated viral transport media using the QIAamp viral ribonucleic acid mini kit method (Qiagen, Los Angeles, CA) according to the manufacturer instructions. All respiratory samples were first tested with universal influenza A and universal influenza B primers and probes. Samples that were positive for universal influenza A were tested with 2009 pandemic influenza A (H1N1) primers and probe sequences. If negative for the pandemic strain, seasonal H1 and H3 specific probes and primer sequences were used. Probes and primers were developed by the US Centers for Disease Control and Prevention, Atlanta, GA. RESULTS Respiratory swab specimens were collected from 426 chil- dren of whom 418 had RTPCR results. Subjects ranged in age from 6 months to 14 years including 233 (56%) participants less than 3 years (Table 1). All subjects agreed to participate. No participants received antiviral medication prior to being tested. About 71% of participants were tested within the first 2 days following symptom onset (range: 0–8 days, median ϭ 2). Among 181 (43%) subjects whose nasal swab was RTPCR-positive for influenza, 142 (78%) were positive for the 2009 pandemic influ- enza A (H1N1) virus and 39 were positive for seasonal influenza viruses (30 type A/H3, 4 type A/H1, and 5 type B). Among the 142 pandemic influenza A (H1N1) RTPCR posi- tive patients, the QuickVue rapid test identified 89 positive tests for a sensitivity of 62.7% (95% confidence interval ͓CI͔: 54.7–70.6). Spec- ificity was 99.2% (95% CI: 98–100). In the 0 to 2 years age group, sensitivity was 76.7% (95% CI: 61.5–91.8). Two false-positives occurred in patients 0 to 2 years of age. The sensitivity of the rapid test was greatest in children less than 3 years of age. In this sample with a pandemic influenza A (H1N1) prevalence of 34%, the rapid test’s Suntarattiwong et al The Pediatric Infectious Disease Journal • Volume 29, Number 4, April 2010 © 2010 Lippincott Williams & Wilkins366 | www.pidj.com
- 4. overall positive predictivevalue was 97.8% and negative predictive value was 81.6%. The sensitivity of the QuickVue test for seasonal influenza viruses was 27/39 or 69.2% (95% CI: 54.7–83.7). RTPCR results obtained from nasal and throat swabs were similar. A total of 142 of 418 (33.97%) nasal swabs were RTPCR positive for 2009 pandemic influenza A (H1N1) virus, while 140 (33.49%) throat swabs were RTPCR positive. The kappa value for agreement between the throat and nasal swab specimens for 2009 pandemic influenza A (H1N1) virus was 0.932 (95% CI: 0.89–0.97). When seasonal influ- enza viruses were included the kappa value was 0.939 (95% CI: 0.91–0.97). DISCUSSION We demonstrated that the QuickVue test had a sensitivity of 62.7% to detect 2009 pandemic influenza A (H1N1) viruses com- pared with RTPCR in febrile pediatric outpatients in Thailand. We used the World Health Organization case definition for ILI. Use of a broader case definition might have influenced the positive and nega- tive predictive values of the rapid test. In contrast to human infections with avian influenza A (H5N1) where throat and lower respiratory tract specimens have been found to be superior to nasal swabs,4 we observed no significant difference between throat and nasal swabs to identify 2009 pandemic influenza A(H1N1) by RTPCR. The performance of RIDTs may vary by virus subtype3 and has been poor when tested with nonhuman influenza viruses.5 While the 2009 pandemic influenza A (H1N1) virus is from a swine lineage, the QuickVue test demonstrated moderate sensitiv- ity similar to the performance of the same test with seasonal influenza viruses in pediatric age groups.6 However, recent small studies have reported lower sensitivity of RIDTs with the 2009 pandemic influenza A (H1N1) virus. Faix et al7 reported that the QuickVue Influenza AϩB test identified 20 of 39 patients who were 2009 pandemic influenza A (H1N1) positive by RTPCR for a sensitivity of 51% (95% CI: 35–67). Vasoo et al8 compared the performance of 3 RIDTs using 60 specimens from children and young adults that were tested positive by Luminex xTAG RVP and reported sensitivity of the QuickVue AϩB test was 53.3% (95% CI: 40.9–65.4). Drexler et al9 reported that compared with RT- PCR, the BinaxNOW Influenza A and B Rapid test had a sensi- tivity of 11.1% (95% CI: 6.7–17.7) in 144 pandemic influenza A(H1N1) positive patients with a median age of 18 years. Several factors may have contributed to the greater sensi- tivity we observed. First, our study population included only children and 71% were tested within 72 hours of symptom onset. Because children shed higher quantities of virus and for longer duration than adults,10 this may explain the higher sensitivity we observed compared with other studies where adult specimens were tested and the time from symptom onset to specimen collection was longer or unknown.7 Previous studies tested specimens that had been shipped, frozen, and thawed prior to testing which may have reduced the performance of the test. In our study, specimen collection was standardized and rapid testing was completed on fresh specimens within minutes of receipt. Seasonal and pandemic viruses continually undergo anti- genic drift which makes regular reassessment of the sensitivity of RIDTs essential. RIDT performance can vary by brand and by virus subtype. Adherence to package insert instructions and careful attention to clinical specimen quality is essential to optimize the performance of all RIDTs. For clinical decision-making, all results from RIDTs must be interpreted in the context of patient’s risk for serious complications, the severity of illness, and circulating in- fluenza strain information. REFERENCES 1. Dawood FS, Jain S, Finelli L, et al. Emergence of a novel swine-origin influenza A (H1N1) virus in humans. N Engl J Med. 2009;360:2605–2615. 2. BOE. Pandemic A (H1N1) update. Thailand Bureau of Epidemiology. 2009. Cited September 3, 2009. Available at: http://203.157.15.4/Flu/ situation/y52/flu_200908310921.pdf. 3. Hurt AC, Alexander R, Hibbert J, et al. Performance of six influenza rapid tests in detecting human influenza in clinical specimens. J Clin Virol. 2007;39:132–135. 4. Abdel-Ghafar AN, Chotpitayasunondh T, Gao Z, et al. Update on avian influenza A (H5N1) virus infection in humans. N Engl J Med. 2008;358: 261–273. 5. Fedorko DP, Nelson NA, McAuliffe J, et al. Performance of rapid tests for detection of avian influenza A virus types H5N1 and H9N2. J Clin Microbiol. 2006;44:1596–1597. 6. Cheng CK, Cowling BJ, Chan KH, et al. Factors affecting QuickVue Influenza AϩB rapid test performance in the community setting. Diagn Microbiol Infect Dis. 2009;65:35–41. 7. Faix DJ, Sherman SS, Waterman SH. Rapid-test sensitivity for novel swine- origin influenza A (H1N1) virus in humans. N Engl J Med. 2009;361:728–729. 8. Vasoo S, Stevens J, Singh K. Rapid antigen tests for diagnosis of pandemic (Swine) influenza A/H1N1. Clin Infect Dis. 2009;49:1090–1093. 9. Drexler JF, Helmer A, Kirberg H, et al. Poor clinical sensitivity of rapid antigen test for influenza A pandemic (H1N1) 2009 virus. Emerg Infect Dis. 2009;15:1662–1664. 10. Steininger C, Kundli M, Aberle SW, et al. Effectiveness of reverse tran- scription-PCR, virus isolation, and enzyme-linked immunosorbent assay for diagnosis of influenza A virus infection in different age groups. J Clin Microbiol. 2002;40:2051–2056. TABLE 1. Sensitivity and Specificity of the QuickVue AϩB Rapid Test Compared to RTPCR Test Age Category* All 0–2 (n ϭ 150) 3–6 (n ϭ 128) 7–14 (n ϭ 100) PCRϩ 30 55 57 142 QVϩ 23 29 37 89 QVϪ 7 26 20 53 Sensitivity (95% CI) 76.7 (61.5–91.8) 52.7 (39.5–65.9) 64.9 (52.5–77.3) 62.7 (54.7–70.6) PCRϪ 120 73 43 237 QVϩ 2 0 0 2 QVϪ 118 73 43 235 Specificity (95% CI) 98.3 (96.0–100) 100 100 99.2 (98.0–100) *Age missing for one child in PCRϪ group. The Pediatric Infectious Disease Journal • Volume 29, Number 4, April 2010 Rapid Influenza Test © 2010 Lippincott Williams & Wilkins www.pidj.com | 367
- 5. SHOULD HIGHER VANCOMYCINTROUGH LEVELS BE TARGETED FOR INVASIVE COMMUNITY-ACQUIRED METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS INFECTIONS IN CHILDREN? Natalia Jimenez-Truque, MQC, MSCI, Isaac Thomsen, MD, Elizabeth Saye, BS, and C. Buddy Creech, MD, MPH Abstract: Methicillin-resistant Staphylococcus aureus isolates with van- comycin minimal inhibitory concentrations (MICs) Ն1.5 g/mL have been associated with poorer clinical outcomes and treatment failures in adults. We evaluated vancomycin MICs in 71 invasive pediatric community- acquired MRSA isolates from 2004 to 2008, using the E-test micromethod and the E-test macro-method. The modal MIC by micromethod was 1.5 g/mL, and median vancomycin MICs did not increase over time. Key Words: MRSA, vancomycin, methicillin resistance, Staphylococcus aureus, pediatric, invasive Accepted for publication October 8, 2009. From the Department of Pediatric Infectious Diseases, Vanderbilt Univer- sity Medical Center, Nashville, TN. Supported, in part, by Vanderbilt CTSA grant 1 UL1 RR024975 from NCRR/NIH and Fogarty International Center grant 1 R25 TW007697. Address for correspondence: C. Buddy Creech, MD, MPH, 1161 21st Ave South, CCC-5311 Medical Center North, Nashville, TN 37232. E-mail: buddy.creech@vanderbilt.edu. DOI: 10.1097/INF.0b013e3181c52a04 Since the emergence of methicillin-resistant S. aureus (MRSA), vancomycin has been a key antimicrobial agent for the treat- ment of MRSA infections.1,2 Concerns surrounding the long-term use of vancomycin as a primary therapy were confirmed when the first vancomycin–intermediate S. aureus (VISA) isolate was diag- nosed in Japan in 1996.3 Recently, it was shown in adult patients that strains with minimal inhibitory concentrations (MICs) Ն1.5 g/mL, though not above the susceptibility breakpoint of 2 g/ mL, were associated with clinical failure.4,5 This increase in vancomycin MICs over time is defined as MIC creep.6 – 8 Because of MIC creep, tissue penetration of vancomycin, and other factors, the vancomycin MIC breakpoints were lowered in 2006. According to these breakpoints, an isolate with an MIC of Յ2 g/mL is considered susceptible to vancomycin, an isolate with intermediate resistance has an MIC of 4 to 8 g/mL, and an isolate with an MIC Ն16 g/mL is resistant. Some bacterial colonies within the staphylococcal population, on exposure to vancomycin, develop an intermediately resistant phenotype known as heterogeneous-vancomycin intermediate S. aureus (hVISA), a phenotype that may be responsible for treatment failures despite overall vancomycin susceptibility.2,6 Most data regarding MIC creep has been collected from adult isolates. It is unclear whether this phenomenon occurs in MRSA isolates from pediatric patients, specifically those classified as community-associated MRSA (CA-MRSA). CA-MRSA iso- lates would be expected to have lower vancomycin MICs when compared with hospital-associated MRSA isolates due to the lack of selective vancomycin pressure in the community. We hypoth- esized that vancomycin MICs have not changed significantly over time in the pediatric population, because risk factors such as frequent vancomycin exposure and foreign bodies such as cathe- ters or prosthetic joints are not likely to be present in children with CA-MRSA disease. To evaluate this, we studied the vancomycin MICs for pediatric CA-MRSA isolates from 2004 to 2008 based on site of infection. MATERIALS AND METHODS S. aureus Clinical Isolates. Since 2004, all pediatric CA-MRSA isolates at Vanderbilt Children’s Hospital have been archived. Each isolate represents a unique pediatric patient. Isolates are considered to be CA-MRSA based on application of Centers for Disease Control-criteria.9 For this study, we analyzed vancomycin MICs for 71 previously characterized invasive pediatric CA- MRSA isolates collected from 2004 to 2008 that were viable in culture and in which site of infection and date were known. These 71 invasive CA-MRSA isolates were randomly selected from a de-identified pediatric clinical isolate database of 1376 unique isolates, using a random number generator. In 706 isolates, unam- biguous notation of site of infection was available; from these, 80 were from patients with invasive MRSA disease, and 71 were viable in culture and had molecular features characteristic of CA-MRSA. Isolates were initially classified as MRSA by the clinical laboratory of Vanderbilt Children’s Hospital and subsequently confirmed by our laboratory based on growth on mannitol salt agar plates containing oxacillin and a positive latex agglutination test for clumping factor (Staphaurex, Remel). DNA was extracted and purified and was used as template for polymerase chain reaction (PCR) detection of nuc and mecA genes and for staphylococcal cassette chromosome mec (SCCmec) typing, as described else- where.10 Genotyping of isolates was performed by pulse-field gel electrophoresis and/or repetitive element sequence based PCR.11 E-Test Micro- and Macro-Methods. S. aureus strain ATCC 29213 was used as the reference strain for both E-test methods (AB- Biodisk, Solna, Sweden), which were performed according to the manufacturer’s guidelines. For the micromethod, a 0.5 McFarland standard was prepared in sterile saline, inoculated onto Mueller- Hinton agar, and incubated for 24 hours at 35°C. For the macro- method, a 2 McFarland standard was inoculated onto brain-heart infusion agar and incubated for 48 hours at 35°C. Testing of the clinical isolates was done in a single laboratory, and the results were recorded by a single observer. S. aureus isolates with van- comycin MICs by micromethod of Յ2 g/mL were considered susceptible (VSSA) based on Clinical and Laboratory Standards Institute guidelines. Intermediate susceptibility to vancomycin (VISA) was defined by MICs of 4 to 8 g/mL, and vancomycin resistance (VRSA) by MICs of Ն16 g/mL. Analysis of MICs over time and by site of infection was performed using the Kruskal-Wallis H method. A P value of 0.05 was considered statistically significant. All analyses were per- formed with SPSS Version 16.0. RESULTS To confirm that each of the 71 isolates clinically determined to be community-associated were also genotypically consistent with CA-MRSA, we performed SCCmec typing and genotyping by pulsed-field gel electrophoresis or rep-PCR. All of the isolates had SCCmec IV cassette and belonged to USA300, the current epi- demic clone in the United States. Of the 71 invasive isolates, 47 (66.2%) were from joint infections (Table 1). Overall, the modal MIC by micromethod was 1.5 g/mL. Fifty-six isolates had an MIC Ն1.5 g/mL. Three isolates had an MIC of 2 g/mL, but are considered susceptible by Clinical and Laboratory Standards Institute breakpoints. Only 15 iso- lates had an MIC Ͻ1.5 g/mL. Median and mean vancomycin MICs did not increase over time (P ϭ 0.245, Inter-Quartile Range [IQR] 1.5–1.5 g/mL). Similarly, MIC values were not significantly different across different infection sites (P ϭ 0.952, IQR ϭ 1.5–1.5 g/mL). Jimenez-Truque et al The Pediatric Infectious Disease Journal • Volume 29, Number 4, April 2010 © 2010 Lippincott Williams & Wilkins368 | www.pidj.com
- 6. By the macro-method,2 isolates (1 from 2005, 1 from 2004) had vancomycin concentrations of 8 and 12 g/ml, respectively. These isolates were susceptible by the micromethod, a character- istic consistent with hVISA. By the macromethod, changes in vancomycin concentrations over time in both the mean and the median were not different (P ϭ 0.052, IQR ϭ 3 to 4 g/mL), nor were the differences by infection sites (P ϭ 0.085, IQR ϭ 3–4 g/mL). DISCUSSION The modal vancomycin MIC of 1.5 g/mL in our isolates is higher than the previously reported modal MIC of 1.0 g/mL for S. aureus.2 This has clinical importance if treatment with vanco- mycin is considered, since MRSA isolates with vancomycin MICs Ն1.5 g/mL have been associated with poorer clinical outcomes and vancomycin treatment failures in adults, despite the fact that they are lower than the vancomycin susceptibility breakpoint.4,5 Though vancomycin is the mainstay of treatment for most children with invasive CA-MRSA infections, the pharmacologic properties of the drug, such as poor penetration into lung and bone12 and potential for nephrotoxicity,12 are challenging. For this reason, many choose clindamycin for first-line therapy for uncom- plicated osteoarticular disease, given the likelihood of susceptibil- ity and excellent bioavailability. However, for bacteremia or com- plicated osteoarticular disease, vancomycin is still recommended by most, and clinicians typically use plasma trough values to evaluate safety and therapeutic window. At our institution, target trough values are set between 5 and 12 g/mL; however, in recent years, many clinicians have pushed the troughs to 10 to 15 g/mL, particularly for osteoarticular disease or pulmonary disease where vancomycin concentrations are only 10% to 15% of plasma.12 In this study, we demonstrated that 56 of 71 isolates had MIC values Ն1.5 g/mL. For patients with bone or joint infections caused by strains with vancomycin MICs of 1.5 g/mL, troughs of no less than 10 g/mL are likely needed for the drug to be fully effective. We did not see a creep in MICs of vancomycin during the period studied. These findings agree with some authors who have reported steady vancomycin MICs over time,2,8 but disagree with others, who have reported the existence of a vancomycin “MIC creep.”6,7 Most of this work has been done in hospital-associated MRSA; therefore, the same selective pressures generating MIC creep are likely not present in this cohort of patients with CA- MRSA. Additionally, by the macromethod, 2 isolates with vanco- mycin concentrations of 8 and 12 g/mL, respectively, are con- sidered to be hVISA. Currently, the E-test macromethod is considered the most sensitive screening method for detecting hVISA.13 Although they represent a low percentage, they might be clinically significant, especially since there are no previous studies that address hVISA in a pediatric population. The 2 hVISA isolates were from years 2004 to 2005, implying that this is not a growing phenomenon in this collection of isolates. One limitation of this study is that the sample size depended on all the available and viable pediatric invasive CA-MRSA isolates at our institution. However, there were no systematic biases towards CA-MRSA isolates with higher vancomycin MICs because until 2009 very few CA-MRSA had formal MIC testing. Another limitation is that we were unable to assess the association between vancomycin MIC and clinical outcomes, since clinical information was unavailable for the patients—a prospective study would be the most definitive way to determine this relation. Last, since many clinical laboratories use the E-test because of its cost-effectiveness, we chose to use this method for MIC determi- nation. While this method can overestimate the MIC when com- pared with broth microdilution,14 the MIC by E-test may be more reliable in predicting vancomycin treatment outcomes.15,16 Clinicians should recognize that MRSA isolates, including those that are epidemiologically and genotypically CA-MRSA, may have higher vancomycin MICs than expected, and that this might complicate response to treatment. We recommend that all pediatric patients treated with vancomycin for invasive CA- MRSA disease, particularly those with osteoarticular disease or pneumonia, have formal MIC testing of their staphylococcal isolate (micromethod) to guide serum vancomycin target trough concentrations. REFERENCES 1. Smith TL, Pearson ML, Wilcox KR, et al. Emergence of vancomycin resistance in Staphylococcus aureus. N Engl J Med. 1999;340:493–501. 2. Jones RN. Microbiological features of vancomycin in the 21st century: minimum inhibitory concentration creep, bactericidal/static activity, and TABLE 1. Median, Mean, Mode, and Range for Vancomycin MICs According to Year and Site of Infection by E-Test Micro- and Macro-Methods N (%) Micro-Method (g/mL) Range Macro-Method (g/mL) RangeMedian (IQR) Mean (95% CI) Mode Median (IQR) Mean (95% CI) Mode Year P ϭ 0.245* P ϭ 0.052* 2004 12 (16.9) 1.5 (1.5–1.5) 1.5 (1.4–1.6) 1.5 1.5–2 5.0 (4–6) 5.3 (3.8–6.9) 6.0 3–12 2005 22 (31.0) 1.5 (1.5–1.5) 1.3 (1.2–1.5) 1.5 1–1.5 4.0 (4–4) 3.9 (3.3–4.4) 4.0 3–8 2006 12 (16.9) 1.5 (1–1.5) 1.4 (1.2–1.5) 1.5 1–1.5 3.5 (3–4) 3.5 (3.2–3.8) 3.0 3–4 2007 15 (21.1) 1.5 (1.5–1.5) 1.4 (1.3–1.5) 1.5 1–1.5 4.0 (3–4) 3.9 (3.4–4.5) 4.0 3–6 2008 10 (14.1) 1.5 (1.5–1.5) 1.5 (1.3–1.7) 1.5 1–1.5 4.0 (3–4) 3.6 (3.2–4.0) 4.0 3–4 Site of infection P ϭ 0.952* P ϭ 0.085* Bone 7 (9.9) 1.5 (1–1.5) 1.4 (1.1–1.6) 1.5 1–1.5 3.0 (3–6) 4.0 (2.7–5.3) 3.0 3–6 Blood 6 (8.5) 1.5 (1.5–1.5) 1.5 (1.5–1.5) 1.5 1.5–1.5 4.0 (3.8–6.5) 4.8 (2.9–6.8) 4.0 3–8 Joint 47 (66.2) 1.5 (1.5–1.5) 1.4 (1.3–1.5) 1.5 1–2 4.0 (3–4) 3.7 (3.5–3.9) 4.0 3–6 CSF 1 (1.4) 1.5 (1.5–1.5) 1.5 (NA) 1.5 1.5–1.5 4.0 (4–4) 4.0 (NA) 4.0 4–4 Pleural fluid 6 (8.5) 1.5 (1–1.6) 1.4 (1–1.8) 1.5 1–2 5.0 (3.8–7.5) 5.8 (2.4–9.2) 4.0 3–12 Pericardial fluid 2 (2.8) 1.5 (1.5–1.5) 1.5 (1.5–1.5) 1.5 1.5–1.5 3.0 (3–3) 3.0 (3–3) 3.0 3–3 Visceral abscess 1 (1.4) 1.5 (1.5–1.5) 1.5 (NA) 1.5 1.5–1.5 4.0 (4–4) 4.0 (NA) 4.0 4–4 Deep tissue abscess 1 (1.4) 1.5 (1.5–1.5) 1.5 (NA) 1.5 1.5–1.5 6.0 (6–6) 6.0 (NA) 6.0 6–6 Total 71 (100) 1.5 (1.5–1.5) 1.4 (1.4–1.5) 1.5 1–2 4.0 (3–4) 4.0 (3.7–4.4) 4.0 3–12 IQR indicates Inter-Quartile Range; 95% CI, 95% Confidence Interval. *Kruskal-Wallis H method. The Pediatric Infectious Disease Journal • Volume 29, Number 4, April 2010 Vancomycin in CA-MRSA © 2010 Lippincott Williams & Wilkins www.pidj.com | 369
- 7. applied breakpoints topredict clinical outcomes or detect resistant strains. Clin Infect Dis. 2006;42:S13–S24. 3. Hiramatsu K, Hanaki H, Ino T, et al. Methicillin-resistant Staphylococcus aureus clinical strain with reduced vancomycin susceptibility. J Antimicrob Chemother. 1997;40:135–136. 4. Sakoulas G, Moise-Broder PA, Schentag J, et al. Relationship of MIC and bactericidal activity to efficacy of vancomycin for treatment of methicillin- resistant Staphylococcus aureus bacteremia. J Clin Microbiol. 2004;42: 2398–2402. 5. Lodise TP, Graves J, Evans A, et al. Relationship between vancomycin MIC and failure among patients with methicillin-resistant Staphylococcus aureus bacteremia treated with vancomycin. Antimicrob Agents Chemother. 2008;52:3315–3320. 6. Wang G, Hindler JF, Ward KW, et al. Increased vancomycin MICs for Staphylococcus aureus clinical isolates from a university hospital during a 5-year period. J Clin Microbiol. 2006;44:3883–3886. 7. Steinkraus G, White R, Friedrich L. Vancomycin MIC creep in non- vancomycin-intermediate Staphylococcus aureus (VISA), vancomycin-sus- ceptible clinical methicillin-resistant S. aureus (MRSA) blood isolates from 2001–05. J Antimicrob Chemother. 2007;60:788–794. 8. Alo´s JI, García-Can˜as A, García-Hierro P, et al. Vancomycin MICs did not creep in Staphylococcus aureus isolates from 2002 to 2006 in a setting with low vancomycin usage. J Antimicrob Chemother. 2008;62:773–775. 9. CDC. Community-Associated MRSA Information for Clinicians. Available at: http://www.cdc.gov/ncidod/dhqp/ar_mrsa_ca_clinicians.html#. Accessed 2005. 10. Oliveira DC, de Lencastre H. Multiplex PCR strategy for rapid identifica- tion of structural types and variants of the mec element in methicillin- resistant Staphylococcus aureus. Antimicrob Agents Chemother. 2002;46: 2155–2161. 11. McDougal LK, Steward CD, Killgore GE, et al. Pulsed-field gel electro- phoresis typing of oxacillin-resistant Staphylococcus aureus isolates from the United States: establishing a national database. J Clin Microbiol. 2003;41:5113–5120. 12. Kollef MH. Limitations of vancomycin in the management of resistant staphylococcal infections. Clin Infect Dis. 2007;45:S191–S195. 13. Wootton M, MacGowan AP, Walsh TR, et al. A multicenter study evalu- ating the current strategies for isolating Staphylococcus aureus strains with reduced susceptibility to glycopeptides. J Clin Microbiol. 2007;45:329– 332. 14. Tenover FC, Lancaster MV, Hill BC, et al. Characterization of Staphylo- cocci with reduced susceptibilities to vancomycin and other glycopeptides. J Clin Microbiol. 1998;36:1020–1027. 15. Hsu DI, Hidayat LK, Quist R, et al. Comparison of method-specific vancomycin minimum inhibitory concentration values and their predictabil- ity for treatment outcome of meticillin-resistant Staphylococcus aureus (MRSA) infections. Int J Antimicrob Agents. 2008;32:378–385. 16. Sader HS, Rhomberg PR, Jones RN. Nine-hospital study comparing broth microdilution and Etest method results for vancomycin and daptomycin against methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother. 2009;53:3162–3165. SURVEILLANCE OF TRANSMITTED RESISTANCE TO ANTIRETROVIRAL DRUG CLASSES AMONG YOUNG CHILDREN IN THE WESTERN CAPE PROVINCE OF SOUTH AFRICA Gert U. van Zyl, MD,* Mark F. Cotton, MD, PhD,† Mathilda Claassen, BSc(Hons),* Charmaine Abrahams, RN,* and Wolfgang Preiser, MD, PhD* Abstract: There are limited data on transmitted antiretroviral resistance in young children who require antiretroviral therapy. We adapted the World Health Organization surveillance strategy, testing antiretroviral naive in- fants (Ͻ18 months) in the Western Cape Province of South Africa, and detecting only 3 non-nucleoside reverse transcriptase inhibitors (NNRTI) and no NRTI or protease inhibitor surveillance mutations in 49 patients. The estimated NRTI and protease inhibitor transmitted antiretroviral resis- tance prevalence is low (Ͻ5%), predicting good therapeutic response in Western Cape infants. Key Words: transmitted antiretroviral drug resistance, surveillance, children, South Africa Accepted for publication October 7, 2009. From the *Division of Medical Virology, Stellenbosch University, Tygerberg, South Africa; and †Department of Paediatrics and Child Health, Children’s Infectious Diseases Clinical Research Unit, Tygerberg Children’s Hospital, Stellenbosch University, Tygerberg, South Africa. Supported by the South African Department of Health, CCMT program. Address for correspondence: Gert U. van Zyl, MD, Division of Medical Virology, Stellenbosch University, Tygerberg Campus, P.O. Box 19063, Tygerberg 7505, South Africa. E-mail: guvz@sun.ac.za. DOI: 10.1097/INF.0b013e3181c4dada Transmitted antiretroviral drug resistance (TDR) refers to the situation when patients are newly infected with antiretroviral resistant HIV strains. In industrialized countries, such as Europe and North America, with about 15 years history of antiretroviral therapy (ART), 5% to 25% of newly HIV-infected individuals are infected with a strain with some degree of antiretroviral drug resistance.1,2 Likewise in developing countries with high levels of antiretroviral exposure, such as Brazil and Argentina, TDR prev- alence of respectively 4% and 7.7% was found.3,4 Transmitted drug resistance testing should ideally be done during the early stages of infection before resistant viruses can revert to wild-type. Since the presence of TDR may compromise the success of ART and therapy roll-out programs, surveillance for prevalence of TDR in populations, where there is increasing ART-usage, is necessary. The World Health Organization (WHO) proposed surveil- lance guidelines using binomial sequential sampling for TDR (threshold surveillance). At most 47 recently infected patients are sequentially tested, allowing prevalence to be classified as either Ͻ5%, between 5% and 15%, or Ͼ15%.5 To gain uniformity and to exclude polymorphic mutations, the list of surveillance drug resis- tance mutations (SDRM) has recently been updated.6 Since ART is rapidly being scaled-up in the Western Cape Province of South Africa, surveillance is needed to detect any increase in TDR. The WHO surveillance strategy targets recently infected adults, but adult surveillance may not accurately predict TDR prevalence in infants, especially where antiretroviral drugs are used for prevention of HIV mother-to-child transmission (PMTCT). The timing of infection is also easier to ascertain in infants than in adults. Transmitted drug resistance data for infants are limited, and for developing countries the data come from PMTCT cohort studies, where high rates of nevirapine (NVP) resistance in infants have been detected.7,8 There are very few infant TDR data from a typical population of children needing ART. In industrialized countries, the TDR prevalence in infants may be high as demonstrated in the United States by Persaud et al who found 5 of 21 (23.8%) infants to have transmitted resistance.9 Recent evidence supports early ART of infants,10 shortly after transmission when TDR may be more significant. Monitoring in young children could provide valuable information for the South African ART roll-out program and enable the selection of optimal regimens. MATERIALS AND METHODS The study formed part of a South African Department of Health funded antiretroviral resistance study that was approved by the Stellenbosch University Committee for Human Research. We adapted the WHO TDR surveillance method to test children, using age, less than 18 months, as indicative of early infection (for adults the cut-off is 3-years after diagnosis, whereas no standard for children has been formulated), applying the same binomial sam- pling method as is recommended for adults. This method is based on lot quality assurance methods. The method does not attempt to van Zyl et al The Pediatric Infectious Disease Journal • Volume 29, Number 4, April 2010 © 2010 Lippincott Williams & Wilkins370 | www.pidj.com
- 8. estimate the exactprevalence but to reliably categorize it: to classify prevalence as either Ͻ5%, between 5% and 15%, or Ͼ15% a sequential sample of at most 47 is needed.5 Genotypic antiretroviral drug resistance testing does not form part of routine management, but patients who were consecutively referred from the Cape Town Metropolitan region were enrolled for baseline genotypic antiretroviral drug resistance testing before initiation of ART from March 2007 to September 2009. About 49 children, from 1 to 17 (median, 3.7) months-of-age, were included, of whom 35 had documented evidence of being exposed to the Western Cape PMTCT- regimen, 6 had no record and 8 did not receive PMTCT. The Western Cape regimen consists of azidothymidine (AZT) from 28 weeks gestation to the mother with additional NVP intrapartum. The baby receives NVP within 72 hours of birth and 7 days of AZT (or 28 days if mothers had less than 4 weeks of AZT). Since there was limited patient information available, we could not access how many patients were fully compliant with the regimen. Antiretroviral resistance testing was done using an accred- ited in-house genotypic sequencing assay. Sequences were inter- preted using the updated WHO Surveillance drug resistance mu- tation list.6 RESULTS Testing yielded 48 reverse transcription and 49 protease sequences. Of these, none had any nucleoside reverse transcriptase inhibitor (NRTI)- or protease inhibitor (PI)- and 3 had non- nucleoside reverse transcriptase inhibitor (NNRTI) SDRM. These 3 patients had received NVP as part of PMTCT. Using the WHO binomial testing model, this survey predicts a low (Ͻ5%) preva- lence of TDR to NRTIs and PIs and, since 3 of the first 47 patients tested had NNRTI resistance, the prevalence of NNRTI resistance can be classified as intermediate: 5% to 15%. DISCUSSION We found a low prevalence (Ͻ5%) of resistance to NRTIs and PIs in antiretroviral naive children whereas 3 out of 48 patients, all of whom had received NVP as part of PMTCT, had NNRTI SDRM. In view of the high NVP exposure through PMTCT regimens, South African children less than 3-years-of-age receive a regimen consisting of NRTIs and a PI. Therefore, we were primarily interested in detecting any transmitted NRTI and PI resistance, while we expected substantial NNRTI resistance. Polymerase chain reaction followed by bulk sequencing is the current standard for TDR surveillance. However, allele-spe- cific assays were shown to detect a higher prevalence of NNRTI mutations in NVP PMTC exposed patients in some studies,11 but not in others.12 Nevertheless, we may have underestimated NNRTI resistance since most NNRTI resistance in these children would be PMTCT-induced, rather than transmitted and competing popula- tions of wild-type virus may more rapidly displace drug-induced mutants, than in the case of transmission of a single resistant strain, where there is lack of competition with wild-type. Bulk sequencing may also underestimate the prevalence of certain transmitted mutations such as M184V due to its effect on viral fitness13 which, although not detected, may result in early virological failure after commencement of therapy.14 The use of these new allele-specific assays has, as yet, not been fully validated in TDR surveillance but we plan to employ them in future studies. The rapid scale-up of ART in South Africa warrants sur- veillance of patient groups in which one can expect to see an increase in the prevalence of transmitted resistance. In the Western Cape Province, especially in urban areas, ART coverage is already high. Using projections from the Actuarial Society of South Africa15 and provincial data (personal communication), the esti- mated ART coverage in April 2009 was 79% for adults and 89% for children; thus, PMTCT coverage is almost universal.16 Some TDR surveillance data for adults exist for sub-Saharan Africa, but worldwide there are few data for children. The choice of ART regimens recommended for children should be carefully consid- ered, especially since therapy needs to be initiated early, and therapy options should be retained to ensure life-long treatment. Based on these data one can expect a good response to the current first-line regimen used in South African children less than 3-years- of-age, which includes NRTIs and a boosted PI. Further TDR surveillance in children, especially in areas such as the Western Cape with high ART and PMTCT coverage, should be a priority and such surveillance should be repeated regularly to detect any increase in prevalence that may compromise antiretroviral regimens. REFERENCES 1. Pillay D. Current patterns in the epidemiology of primary HIV drug resistance in North America and Europe. Antivir Ther. 2004;9:695–702. 2. Chaix ML, Descamps D, Wirden M, et al. Stable frequency of HIV-1 transmitted drug resistance in patients at the time of primary infection over 1996–2006 in France. AIDS. 2009;23:717–724. 3. Petroni A, Deluchi G, Pryluka D, et al. Update on primary HIV-1 resistance in Argentina: emergence of mutations conferring high-level resistance to nonnucleoside reverse transcriptase inhibitors in drug-naive patients. J Acquir Immune Defic Syndr. 2006;42:506–510. 4. Rodrigues R, Scherer LC, Oliveira CM, et al. Low prevalence of primary antiretroviral resistance mutations and predominance of HIV-1 clade C at polymerase gene in newly diagnosed individuals from south Brazil. Virus Res. 2006;116:201–207. 5. Myatt M, Bennett DE. A novel sequential sampling technique for the surveillance of transmitted HIV drug resistance by cross-sectional survey for use in low resource settings. Antivir Ther. 2008;13(suppl 2):37–48. 6. Bennett DE, Camacho RJ, Otelea D, et al. Drug resistance mutations for surveillance of transmitted HIV-1 drug-resistance: 2009 update. PLoS ONE. 2009;4:e4724. 7. Church JD, Mwatha A, Bagenda D, et al. In utero HIV infection is associated with an increased risk of nevirapine resistance in Ugandan infants who were exposed to perinatal single dose nevirapine. AIDS Res Hum Retroviruses. 2009;25:673–677. 8. Arrive E, Newell ML, Ekouevi DK, et al. Prevalence of resistance to nevirapine in mothers and children after single-dose exposure to prevent vertical transmission of HIV-1: a meta-analysis. Int J Epidemiol. 2007;36: 1009–1021. 9. Persaud D, Palumbo P, Ziemniak C, et al. Early archiving and predomi- nance of nonnucleoside reverse transcriptase inhibitor-resistant HIV-1 among recently infected infants born in the United States. J Infect Dis. 2007;195:1402–1410. 10. Violari A, Cotton MF, Gibb DM, et al. Early antiretroviral therapy and mortality among HIV-infected infants. N Engl J Med. 2008;359:2233– 2244. 11. Hauser A, Mugenyi K, Kabasinguzi R, et al. Detection and quantification of minor human immunodeficiency virus type 1 variants harboring K103N and Y181C resistance mutations in subtype A and D isolates by allele-specific real-time PCR. Antimicrob Agents Chemother. 2009;53:2965–2973. 12. Church JD, Huang W, Parkin N, et al. Comparison of laboratory methods for analysis of non-nucleoside reverse transcriptase inhibitor resistance in Ugandan infants. AIDS Res Hum Retroviruses. 2009;25:657–663. 13. Toni TA, Asahchop EL, Moisi D, et al. Detection of human immunodefi- ciency virus (HIV) type 1 M184V and K103N minority variants in patients with primary HIV infection. Antimicrob Agents Chemother. 2009;53:1670– 1672. 14. Metzner KJ, Giulieri SG, Knoepfel SA, et al. Minority quasispecies of drug-resistant HIV-1 that lead to early therapy failure in treatment-naive and -adherent patients. Clin Infect Dis. 2009;48:239–247. 15. Dorrington R. Western Cape HIV ASSA projection output. 2005. Available at: http://www.capegateway.gov.za/eng/pubs/reports_research/W/142134. Accessed June 23, 2009. 16. Eley B. Addressing the paediatric HIV epidemic: a perspective from the Western Cape Region of South Africa. Trans R Soc Trop Med Hyg. 2006;100:19–23. The Pediatric Infectious Disease Journal • Volume 29, Number 4, April 2010 Antiretroviral Resistance © 2010 Lippincott Williams & Wilkins www.pidj.com | 371
- 9. MULTIMETHOD ADHERENCE ASSESSMENTIN CHILDREN WITH PERINATALLY ACQUIRED HIV-1 THE INFLUENCE OF OFF-SCHEDULE DOSING IN PREDICTING BIOLOGICAL MARKERS Patricia A. Garvie, PhD,*† Megan L. Wilkins, PhD,* Elizabeth D. Kolivas, BA,* and J. Christopher Young, MA* Abstract: To improve upon adherence assessment in children with HIV, multimethod adherence strategies (pill count, missed doses, off-schedule dosing) were conducted concurrent with viral load and CD4% biomarker assays. Off-schedule dosing predicted both health status markers, while the more common strategies did not. Findings support inclusion of off- schedule dosing concurrent with collection of biomarkers to assess adher- ence in children with HIV. Key Words: adherence assessment, off-schedule dosing, pediatric HIV, youth, ARV Accepted for publication October 16, 2009. From the *Department of Behavioral Medicine, St. Jude Children’s Research Hospital, Memphis, TN; and †Department of Pediatrics, University of Tennessee Health Science Center, Memphis, TN. Supported by the American Lebanese Syrian Associated Charities. The funding source had no role or input in the design or conduct of the study; collection, management, analysis, or interpretation of the data; or preparation, review, or approval of the manuscript. The authors have no conflicts of interest or financial disclosures to make. Address for correspondence: Patricia A. Garvie, PhD, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, MS 740, Memphis, TN 38105. E-mail: patti.garvie@stjude.org. DOI: 10.1097/INF.0b013e3181c67686 Medication adherence and accurate adherence assessment in children with HIV are vital to effective treatment. Nonad- herence to antiretroviral (ARV) medications allows viral replica- tion and mutation that can lead to treatment resistance and more complicated medication regimens.1 No single best adherence as- sessment strategy exists for children with HIV. Utilizing multi- method assessment strategies may better capture adherence behav- iors and address limitations associated with any single measure. While pill counts yield significant associations with viral load (VL),2– 6 clinical utility is limited by significant staff time demands, cost constraints, and potential social desirability (eg, “pill dumping”).7 Retrospective recall is the most commonly used adherence assessment strategy because of ease of administration, cost effectiveness, and clinical utility.7 Recall methods typically rely on caregiver report of how many doses were missed within a specified period. Methodologic limitations, including socially de- sirable responding, difficulty recalling everyday events, and lack of continuous assessment may account for inconsistent findings with VL2,3 and may result in adherence overestimations than object- ive methods.7 Recent pediatric studies failed to find a relationship between caregivers’ 3-day recall of missed doses and child VL2 while others reported significant findings with VL.3 Inconsistent reports necessitate further investigation into the accuracy and efficacy of adherence recall methods for children with HIV. Dose timing is an essential aspect of adherence due to risk of developing medication resistance associated with temporal dosing inconsistency.1 To better evaluate relationships among adherence with VL and CD4 biomarkers, off-schedule dosing should be assessed. True adherence may be overestimated when doses are not missed, but still not taken as prescribed.4,8 This study contributes to better understanding the impact of off-schedule dosing on child health outcomes. Treatment efficacy and disease progression are monitored routinely via CD4 and VL biomarker assays. Within the pediatric HIV adherence literature, investigations frequently only reported VL.2,4,6,8,9 Given the contribution of each biomarker to under- standing disease progression and treatment response, concurrent CD4 and VL monitoring is warranted. Reported relationships among CD4 and adherence in children with HIV have been mixed,3,5,10 and comparison of findings is complicated by whether CD4 count, CD4%, or both were monitored. Biomarkers typically have not been monitored concurrently with adherence, often collected within 90 days of adherence assess- ment.2,6 Studies utilizing concurrent assessment reliably detect sig- nificant VL-adherence relationships.3,4,9,10 Concurrent CD4-adher- ence assessments are limited and findings are mixed.3,5,10 To improve on prior methodology, the present study col- lected VL and CD4% biomarkers concurrent with multimethod adherence assessment strategies. Off-schedule dosing was mea- sured to investigate the role of inconsistent interval dosing on child VL and CD4%. Hypotheses: (1) pharmacy pill count would predict both child VL and CD4%; and (2) caregiver report of 3-day missed and 3-day off-schedule dosing would predict both child increased VL and decreased CD4%. METHOD Participants Sixty caregivers of children with perinatally acquired HIV-1 presenting for routine multidisciplinary pediatric HIV care in the Mid-Southern United States were recruited consecutively. Child demographics: Mean (M) age 8.0 years (SD ϭ 4.26); 53% female; 85% African American, 13% Caucasian; 92% were on a twice daily, 3-drug (75%) or 4-drug regimen (17%). Caregiver demo- graphics: Mean age 39.7 years (SD ϭ 11.6); 93% women; 85% African American, 15% Caucasian; 66.7% biologic parent, 25% other relative, 8% adoptive parent; 55% HIV-positive; 57% un- employed. Data Collection This single-site prospective cross-sectional study was insti- tutional review board-approved. Caregivers provided written con- sent, and completed a study-specific semistructured interview to obtain demographic information and caregiver report of child medication adherence via 3-day retrospective recall of number of child ARV doses missed, and doses taken, but off-schedule (not as prescribed). Additionally, ARV adherence was assessed by phar- macy pill count (percentage of medication returned/total medica- tion dispensed). Child CD4 and VL assays were drawn per routine clinical care on the date of caregiver participation, and pharmacy pill count was obtained. One critical aspect of the 3-day recall methodology imple- mented includes calculations of off-schedule dosing in relation to reported missed doses. Participants were asked at what time(s) the child was required to take ARV medications and which medica- tions were prescribed for each dose time, allowing for the assess- ment of medications prescribed once-daily within a twice-daily regimen. Respondents were asked “how many doses of each ARV medication were missed yesterday morning? And, evening?” “How about the day before that?” “And, the day before that?” Similarly, to assess off-schedule dosing, caregivers were asked, “Over the past 3 days, how many doses of each ARV medication was not taken when it was supposed to be?” beginning with yesterday morning, etc. Caregivers estimated by how much time each off-schedule dose was taken early or late. Doses taken within 1 hour of the expected dose time were considered adherent. Off-schedule Garvie et al The Pediatric Infectious Disease Journal • Volume 29, Number 4, April 2010 © 2010 Lippincott Williams & Wilkins372 | www.pidj.com
- 10. calculations took intoaccount doses missed, determining the propor- tion of doses actually taken, but not as prescribed. Statistical Analyses Analyses were conducted using SPSS 15.0. Due to signifi- cant skewness, variables were dichotomized based on clinical criteria. Child CD4% was recoded by Center for Disease Control clinical classification of immunosuppression (Ͻ25% vs. Ն25%). VL was dichotomized as undetectable/detectable (Ͻ400 copies/mL vs. Ն400 copies/mL). Caregiver report of missed doses and off- schedule dosing were recoded as nonadherent/adherent (missed vs. none; off-schedule vs. none). Pharmacy pill counts were dichoto- mized (Ͻ93% vs. Ն93%) corresponding with missing Ͼ2 days’ doses within 30 days. Phi coefficient analyses examined relationships among the 3 adherence assessment strategies. Predictive validity of each assessment strategy on child CD4% and VL was analyzed via logistic regression. RESULTS Adherence by pharmacy pill count was M ϭ 91.41% (SD ϭ 9.71), and Ͼ95% for 40% of the sample. Further, 83% of care- givers reported no missed doses for their child over the 3-day recall period; 77% reported no off-schedule doses during the same 3-day period. Caregivers reported ϳ5.7% of doses were missed (Median ͓Mdn͔ ϭ 0, SD ϭ 17.49) and 12% taken off-schedule (Mdn ϭ 0, SD ϭ 26.87); thus, ϳ18% (Mdn ϭ 0, SD ϭ 29.95) of all prescribed doses were either missed or taken off-schedule per 3-day recall. Dichotomously coded adherence variables were evaluated for multicollinearity. Results (Table 1) revealed a significant relationship between adherence assessed by pill count and 3-day recall of doses missed ( ϭ Ϫ0.29, P ϭ 0.02). Neither pharmacy pill count ( ϭ Ϫ0.05, P ϭ 0.69) nor 3-day recall of doses missed significantly related to doses taken off-schedule ( ϭ 0.18, P ϭ 0.17). Univariate logistic regression analyses (Table 2) revealed caregiver 3-day recall of off-schedule doses significantly predicted child CD4% suppression (Wald 2 ϭ 4.44, P ϭ 0.04, OR ϭ 4.18; CI0.95 ϭ 1.11–15.79), but caregiver report of missed doses (Wald 2 ϭ 0.48, P ϭ 0.49, OR ϭ 1.71; CI0.95 ϭ 0.38–7.84) and pharmacy pill count (Wald 2 ϭ 0.40, P ϭ 0.53, OR ϭ 1.53; CI0.95 ϭ 0.41–5.68) did not. Caregiver report of doses taken off-schedule (Table 3) significantly predicted child detectable VL (Wald 2 ϭ 3.64, P ϭ 0.06, OR ϭ 3.38; CI0.95 ϭ 0.97–11.78), but doses missed (Wald 2 ϭ 0.66, P ϭ 0.42, OR ϭ 0.55; CI0.95 ϭ 0.13–2.36) and pharmacy pill count (Wald 2 ϭ 1.67, P ϭ 0.20, OR ϭ 0.50; CI0.95 ϭ 0.17–1.43) did not. DISCUSSION Validating ARV adherence assessment remains of para- mount importance, both in pediatric HIV clinical care and clinical investigation. Nonetheless, a gold standard to assess adherence remains elusive. Study findings emphasize the importance of including off-schedule dosing assessment when evaluating adher- ence. Results also support inclusion of both CD4% and VL biomarkers given independent relationships to self-reported adher- ence. Collecting both indices, rather than VL alone, increased the likelihood of identifying adherence difficulties in children with perinatally acquired HIV and the impact of suboptimal adherence on both immune composition and viral control. The most commonly used assessment strategies, pharmacy pill count and caregiver recall of missed doses, failed to predict child VL or CD4% suppression, despite relatively high adherence rates. Inclusion of off-schedule dosing provided a more refined analysis of overall adherence behaviors. Participants who other- wise appeared adherent by pill count and/or doses missed ac- knowledged difficulty administering medications as prescribed, an extremely important health consideration given risk to develop medication resistance because of temporally inconsistent ARV dosing. Thus, off-schedule dosing assessment may represent a more externally valid and sensitive measure of adherence behav- ior, providing clinical insight beyond other methods. Study findings are tempered by limitations inherent to a relatively small 1-time cross-sectional single-site sample, and utilization of self-report measures and pill counts, which are vulnerable to social desirability demands. Dichotomization of data due to skewness also may limit findings. Limited methodologic and analytical descriptions in prior studies compounded by contradictory results make interpretation, comparison across studies, and replication difficult. Study findings demonstrate concurrent assessment of biomarkers and off-sched- ule dosing more accurately illulminate the influence of nonadher- ence behaviors in children with HIV. Routinely employing these methods may allow earlier detection and intervention to improve dose-timing consistency and prevent viral replication and devel- opment of medication resistance. Methodologic detail regarding how adherence is assessed and analyzed should be included in future reports to encourage replication and permit comparison across studies. Replication with a larger multisite cohort across treatment settings to clarify further the contribution of off-schedule dosing, concurrent collection of biologic assays, and adherence assessment is warranted. ACKNOWLEDGMENTS The authors thank the families living with HIV who partic- ipated and whose contributions provided invaluable information TABLE 1. Phi Coefficient Correlations Between Multiple Measures of Adherence Pill Count Recall Missed Recall Off-Schedule Pharmacy pill count — Ϫ0.29* Ϫ0.05 Recall missed — 0.18 Recall off-schedule — *P ϭ 0.02. TABLE 2. Prediction of CD4 Suppression (CD4% Ͻ25) Wald OR CI0.95 Total recall adherence 5.43* 4.67 1.28–17.28 Recall off-schedule 4.44† 4.18 1.11–15.80 Recall missed doses 0.48 1.71 0.38–7.84 Pharmacy pill count 0.40 1.53 0.41–5.68 *P ϭ 0.02. † P ϭ 0.03. TABLE 3. Prediction of Detectable Viral Load (Ͼ400) Wald OR CI0.95 Total recall adherence 0.85 1.67 0.56–4.93 Recall off-schedule 3.64* 3.38 0.97–11.78 Recall missed doses 0.66 0.55 0.13–2.36 Pharmacy pill count 1.67 0.50 0.17–1.43 *P ϭ 0.06. The Pediatric Infectious Disease Journal • Volume 29, Number 4, April 2010 Off-Schedule Dosing in HIV © 2010 Lippincott Williams & Wilkins www.pidj.com | 373
- 11. about their experienceswith medication adherence for their chil- dren; graduate research assistants who contributed toward data collection and entry, Will Dalton, III, PhD, Rebecca West De- deaux, MS, Ericka Midgett, MS, Christy Jayne Curtis, PhD, and Clinical Research Associates, Jo Lawford, PhD, and Megan Ba- net, MA, who in addition monitored data collection, developed the database, and managed the data. REFERENCES 1. Liu H, Miller LG, Hays RD, et al. Repeated measures longitudinal analyses of HIV virologic response as a function of the percent adherence, dose timing, genotypic sensitivity and other factors. J Acquir Immune Defic Syndr. 2006;41:315–322. 2. Farley J, Hines S, Musk A, et al. Assessment of adherence to antiviral therapy in HIV-infected children using the medication event monitoring system, pharmacy refill, provider assessment, caregiver self-report, and appointment keeping. J Acquir Immune Defic Syndr. 2003;33:211–218. 3. Van Dyke RB, Lee S, Johnson GM, et al. Reported adherence as a determinant of response to highly active antiretroviral therapy in children who have human immunodeficiency virus infection. Pediatrics. 2002;109:e61. 4. Wiener L, Riekert K, Ryder C, et al. Assessing medication adherence in adolescents with HIV when electronic monitoring is not feasible. AIDS Patient Care STDS. 2004;18:527–538. 5. Watson DC, Farley JJ. Efficacy of and adherence to highly active antiret- roviral therapy in children infected with human immunodeficiency virus type 1. Pediatr Infect Dis J. 1999;18:682–689. 6. Marhefka SL, Farley JJ, Rodrigue JR, et al. Clinical assessment of medi- cation adherence among HIV-infected children: examination of the treat- ment interview protocol (TIP). AIDS Care. 2004;16:323–338. 7. Kerr T, Walsh J, Lloyd-Smith E, et al. Measuring adherence to highly active antiretroviral therapy: implications for research and practice. Curr HIV/ AIDS Rep. 2005;2:200–205. 8. Marhefka SL, Tepper VJ, Farley JJ, et al. Brief report: assessing adherence to pediatric antiretroviral regimens using the 24-hour recall interview. J Pediatr Psychol. 2006;31:989–994. 9. Naar-King S, Frey M, Harris M, et al. Measuring adherence to treatment of paediatric HIV/AIDS. AIDS Care. 2005;17:345–349. 10. Martin S, Elliot-DeSorbo DK, Wolters PL, et al. Patient, caregiver, and regimen characteristics associated with adherence to highly active antiret- roviral therapy among HIV-infected children and adolescents. Pediatr Infect Dis J. 2007;6:61–67. POPULATION BASED EXTERNAL VALIDATION OF A EUROPEAN PREDICTIVE MODEL FOR RESPIRATORY SYNCYTIAL VIRUS HOSPITALIZATION OF PREMATURE INFANTS BORN 33 TO 35 WEEKS OF GESTATIONAL AGE Lone G. Stensballe, MD, PhD,* John R. Fullarton, PhD,† Xavier Carbonell-Estrany, MD,‡ and Eric A. F. Simo˜es, MD§ Abstract: Prospectively collected population-based data on 2529 Danish infants born at 33 to 35 weeks of gestation were used to validate an European predictive model of respiratory syncytial virus (RSV) hospital- ization. The model was found to be robust with a diagnostic accuracy of 65.9% to distinguish between RSV-hospitalized versus non-RSV-hospital- ized Danish infants born at 33 to 35 weeks of gestation. Accepted for publication October 27, 2009. From the *Bandim Health Project, Statens Serum Institut, Copenhagen, Denmark; †Strategen Limited, Basingstoke, Hampshire, United Kingdom; ‡Neonatology Service, Hospital Clinic, Institut Clinic de Ginecologia Obstetri- cia I Neonatologia, Agrupacio´ Sanitaria Hospital Clínic-Hospital SJ Deu, Universitat de Barcelona, Barcelona, Spain; and §Department of Pediatrics, Section of Infectious Diseases, The University of Colorado School of Medicine and The Children’s Hospital, Denver, CO. Supported by Abbott Laboratories for work on various projects (to J.R.F.). Also, by the Abbott Laboratories (to L.G.S., E.S., and X.C.E.). The data management for the establishment of the Danish dataset was funded un-restricted by Abbott Laboratories, Denmark. Address for correspondence: Lone G. Stensballe, MD, PhD, Bandim Health Project, Statens Serum Institut, Copenhagen, Denmark. E-mail: lgn@ssi.dk. DOI: 10.1097/INF.0b013e3181c810da Premature infants are at increased risk of hospitalization, with severe respiratory syncytial virus (RSV) airway infection.1 Passive immunoprophylaxis with a humanized monoclonal anti- body, palivizumab, has been shown to reduce the risk of RSV hospitalization in preterm infants.2 However, passive immunopro- phylaxis for all infants born 33 to 35 weeks of gestation (wGA) is not considered cost-effective in most European countries.3 Thus, there is a need, in an evidence-based manner, to identify infants born 33 to 35 wGA with the greatest likelihood of RSV hospital- ization in whom to target immunoprophylaxis. This approach has recently been modeled by Simoes et al4,5 who used data on infants born 33 to 35 wGA from Spain and Germany to define a European RSV hospitalization predictive model. However the rates of RSV hospitalization in Northern Europe are lower than the rates in Spain, prompting us to examine the usefulness of this model in a Scandinavian population. We used population-based data prospectively collected on 2529 infants born 33 to 35 wGA in Denmark to present an external statistical test of the robustness of the European RSV predictive model. METHODS In the publication by Simoes et al,5 7 variables describing the most important risk factors for RSV hospitalization in prema- ture infants 33 to 35 wGA in Spain were identified. These variables were “birth within 10 weeks of the start of the season,” “birth weight,” “breast-feeding Յ2 months,” “number of siblings Ն2 years of age,” “number of family members with atopy,” “male sex,” and “number of family members with wheeze.” In the original study, information on these 7 factors examined by dis- criminant function analysis resulted in a diagnostic accuracy of 71% when trying to identify premature infants hospitalized with RSV. Discriminant function analysis optimizes the variables which discriminate between 2 or more naturally occurring groups, here RSV-hospitalized versus non-RSV-hospitalized premature infants 33 to 35 wGA. Using prospectively collected population based data on 93,620 births during 1997 to 2003 from the Danish National Birth Cohort (DNBC) (www.BSMB.dk),6 2529 infants born 33 to 35 wGA and followed until the age of 18 months after birth were identified and data on the 7 variables described above were withdrawn in August 2008. For 3 of the 7 variables, the DNBC data did not exactly represent the Spanish data. In the Spanish data, information on siblings was defined to be number of siblings Ն2 years of age, but in the DNBC data number of siblings represented the total number of siblings below 12 years of age excluding any twin from multiple births. In the Spanish data, information on close relatives with wheeze or atopy included information on siblings and grandparents; but in the DNBC data close relatives with atopy was calculated with no data on grandparents available, and close relatives with wheeze was calculated with no data on grandparents and siblings available. Furthermore, other variables containing information on other established RSV hospitalization risk factors and information on mortality were withdrawn from DNBC. The additional risk factors included data on parental smoking during and after pregnancy, parental educational level, pets in the home, day care attendance, and chronic lung disease. The information on RSV season also included in the modeling was based on the Danish RSV season, November to April. Stensballe et al The Pediatric Infectious Disease Journal • Volume 29, Number 4, April 2010 © 2010 Lippincott Williams & Wilkins374 | www.pidj.com
- 12. This Danish datasetwas used to externally validate the robustness of the European Predictive Model. Information on the 7 variables in the Danish data was used to generate a discriminant function in the Danish dataset itself, resulting in a percentage value of true classification (here, of predicted RSV hospitalization) and receiver-operating-characteristic (ROC) plots of the sensitivity by (1–specificity) with areas closest to 1 predicting best predictive accuracy. Premature 33 to 35 wGA case infants who experienced RSV hospitalization were compared with premature 33 to 35 wGA case infants without RSV hospitalization. The analysis was carried out on all data with automatic substitution of neutral values for missing data, and secondarily, with exclusion of missing values. The neutral values were calculated from the mean values in the 2 outcome groups (hospitalized and nonhospitalized) and set so as to provide no bias in the discriminate functions derived from the data so modified. There were complete information on date of birth, sex, siblings, and on if any family member among parents or siblings had any atopic disease. For information on if any family member had wheeze, 32 individuals (1.3%) had missing informa- tion. For birth weight, 46 individuals (1.8%) had missing infor- mation. For breast-feeding, 993 individuals (39.3%) had missing information. The model was sought to be improved by serially removing variables from the model, by inclusion on information on the other established risk factors, and by substituting categorical (Yes/No) variables for atopy and wheeze. Finally, the unadjusted coefficients generated from the Spanish study were applied to the Danish data. All analyses were carried out using SPSS 15 for Windows. RESULTS A total of 2614 infants included in DNBC were born 33 to 35 wGA (gestational days 231 to 251). Of them, 37 stillborn infants were excluded. Forty-six infants who died during fol- low-up were excluded from the analyses, one had chronic lung disease and none of these infants experienced RSV hospitaliza- tion. 2 infants without RSV hospitalization had chronic lung disease and were excluded from the analyses. Of the 2529 infants born 33 to 35 wGA left for follow-up, 139 (5.5%) experienced an RSV hospitalization. When the Danish data were used to validate the 7-variable European Predictive Model and the analysis was carried out on all data with automatic substitution of neutral values for missing data, the resulting diagnostic accuracy was 65.9%, with an area under the ROC curve of 0.625 (0.578–0.673) (Figure 1). The negative predictive value at the point of maximum discrimination for the Danish data was 0.86. If the missing data was excluded from the analyses, the resulting diagnostic accuracy was 63.3%, with an area under the ROC curve of 0.598 (0.524–0.671). The exclusion of missing data reduced the number of RSV cases to 66 and controls to 991. When variables were serially removed from the model, improvement was achieved when atopy was eliminated, giving 68.1% correct classification and AUC 0.634 (0.586–0.681). Op- timization was attempted by substituting categorical (Yes/No) variables for atopy and wheeze and by using all the variables in the data set, but with no improvement. Since the variable “birth within 10 weeks of the start of the season” had the greatest positive impact on the fit of the predictive model, further optimization was achieved by stratifying the data by season, leading to improvement of the diagnostic accuracy in some but not all seasons (data not shown, available on request). When the discriminant function analysis was based on the unadjusted coefficients from the FLIP dataset with information on Spanish infants 33 to 35 wGA, the diagnostic accuracy was 62% and the area under the ROC curve was 0.598 (0.553–0.644). DISCUSSION The present population-based cohort study externally vali- dated the recently published European RSV hospitalization pre- dictive study to test the robustness of that model when used in other European infant populations. The model included informa- tion on globally established risk factors for RSV hospitalization: time of birth, birth weight, male sex, breast-feeding, siblings, and family disposition to atopy and wheeze.1,7,8 Using data on Danish premature infants born week 33 to 35 of gestation, we found a diagnostic accuracy of nearly 66% of the model. A diagnostic accuracy of about 66% might not appear high. However, in the study by Simoes et al5 where the original model tested in the present study was generated it was found that using, for example, the Spanish Guidelines to identify premature children at increased risk of RSV hospitalization was not better than chance. Denmark has no specific guidelines; the Danish Pediatric Society recommends individual judgment when the use of RSV prophylaxis is considered. Our study was limited by several factors. Despite the large overall sample size, the total number of 139 RSV hospitalized infants born 33 to 35 wGA set statistical limitations. The data set were not precisely alike the data collected for the FLIP study and some of the variables had considerable missing values. We tested the diagnostic accuracy in the subset of the DNBC dataset without any missing information and found it to be essentially the same. FIGURE 1. ROC curve: Receiver-operating-characteristic (ROC) plot (sensitivity by (1–specificity)) for the Danish 7 variable model with automatic substitution of neutral val- ues for missing data. Notes: Data from the Danish National Birth Cohort (DNBC) (www.BSMB.dk) August 2008. The 7 variables were “birth within 10 weeks of the start of the season,” “birth weight,” “breast-feeding Յ2 months,” “number of siblings Ն2 years of age,” “number of family members with atopy,” “male sex,” and “number of family members with wheeze.” ROC plot areas closest to 1 predict best pre- dictive accuracy. The Pediatric Infectious Disease Journal • Volume 29, Number 4, April 2010 RSV Hospitalization Predictive Model © 2010 Lippincott Williams & Wilkins www.pidj.com | 375
- 13. However, the EuropeanRSV hospitalization predictive model proved robust to external validation and the diagnostic accuracy remained satisfying. Recently, such a RSV risk-scoring tool based on information on the same 7 RSV risk factors was evaluated in Canadian infants 33 to 35 wGA and was found to be practical and effective in reducing RSV hospitalization in infants who are most at risk, while avoiding prophylaxis in the 82% of the GA cohort who were considered low risk for RSV infection.9 In countries like Denmark without RSV prophylaxis guidelines for premature infants and where the use of RSV prophylaxis is based solely on individual judgment, a risk-scoring model might improve the use and efficacy of RSV prophylaxis. CONCLUSION AND PERSPECTIVE This external validation of the European RSV predictive model confirmed the robustness of the model. Based on the model, computer software could be developed and offered to European neonatal de- partments to optimize the cost-effectiveness of the use of passive prophylaxis against RSV in premature infants born 33 to 35 wGA. REFERENCES 1. Law BJ, Langley JM, Allen U, et al. The Pediatric Investigators Collab- orative Network on Infections in Canada study of predictors of hospital- ization for respiratory syncytial virus infection for infants born at 33 through 35 completed weeks of gestation. Pediatr Infect Dis J. 2004;23: 806–814. 2. Connor EM; The IMpact-RSV Study Group. Palivizumab, a humanized respiratory syncytial virus monoclonal antibody, reduces hospitalization from respiratory syncytial virus infection in high-risk infants ͓see com- ments͔. Pediatrics. 1998;102:531–537. 3. Wang D, Cummins C, Bayliss S, et al. Immunoprophylaxis against respira- tory syncytial virus (RSV) with palivizumab in children: a systematic review and economic evaluation. Health Technol Assess. 2008;12:iii, ix–x, 1–86. 4. Figueras-Aloy J, Carbonell-Estrany X, Quero J. Case-control study of the risk factors linked to respiratory syncytial virus infection requiring hospital- ization in premature infants born at a gestational age of 33–35 weeks in Spain. Pediatr Infect Dis J. 2004;23:815–820. 5. Simoes EA, Carbonell-Estrany X, Fullarton JR, et al. A predictive model for respiratory syncytial virus (RSV) hospitalization of premature infants born at 33–35 weeks of gestational age, based on data from the Spanish FLIP Study. Respir Res. 2008;9:78. 6. Olsen J, Melbye M, Olsen SF, et al. The Danish National Birth Cohort— its background, structure and aim. Scand J Public Health. 2001;29:300– 307. 7. Stensballe LG, Kristensen K, Simoes EA, et al. Atopic disposition, wheez- ing, and subsequent respiratory syncytial virus hospitalization in Danish children younger than 18 months: a nested case-control study. Pediatrics. 2006;118:e1360–e1368. 8. Carbonell-Estrany X, Figueras-Aloy J, Law BJ. Identifying risk factors for severe respiratory syncytial virus among infants born after 33 through 35 completed weeks of gestation: different methodologies yield consistent findings. Pediatr Infect Dis J. 2004;23:S193–S201. 9. Paes B, Steele S, Janes M, et al. Risk-scoring tool for respiratory syncytial virus prophylaxis in premature infants born at 33–35 completed weeks’ gestational age in Canada. Curr Med Res Opin. 2009;25:1585–1591. ANTIRETROVIRAL-RELATED HEMATOLOGIC SHORT-TERM TOXICITY IN HEALTHY INFANTS IMPLICATIONS OF THE NEW NEONATAL 4-WEEK ZIDOVUDINE REGIMEN Rebeca Lahoz, MD,* Antoni Noguera, MD, PhD,* Nu´ria Rovira, MD,* Albert Catala`, MD,* Emília Sa´nchez, MD, PhD,† Rafael Jime´nez, MD, PhD,* and Cla`udia Fortuny, MD, PhD* Abstract: Recent updates of the guidelines on the prevention of human immunodeficiency virus mother-to-child transmission have shortened the neonatal zidovudine prophylactic regimens from 6 to 4 weeks. We present a prospective observational study in a large cohort of mother-infant pairs and report that the 4-week regimen allows an earlier recovery of the anemia in these otherwise healthy infants. Key Words: hematologic toxicity, human immunodeficiency virus, neonatal prophylaxis, pediatrics, zidovudine Accepted for publication October 27, 2009. From the Departments of *Pediatrics, and †Hematology, Pediatric Infectious Diseases Unit, Hospital Sant Joan de De´u-Universitat de Barcelona, Barce- lona, Spain; and ‡Catalan Agency for Health Technology Assessment and Research, Barcelona, Spain. Address for correspondence: Antoni Noguera, MD, PhD, Infectious Diseases Unit, Pediatrics Department, Hospital Sant Joan de De´u, Passeig Sant Joan de De´u 2, 08950 Esplugues, Barcelona, Spain. E-mail: ton@hsjdbcn.org. Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s Web site (www.pidj.com). DOI: 10.1097/INF.0b013e3181c81fd4 Mother-to-child transmission (MTCT) of human immuno- deficiency virus (HIV) in developed countries has de- creased to Ͻ2% after the implementation of prophylactic mea- sures, such as the use of antiretrovirals (ARV), elective cesarean section, and refraining from breast-feeding.1–3 Expo- sure of these otherwise healthy infants to one or more drugs of unknown toxicity is of concern. To date, most of the reported hematologic and mitochondrial adverse effects, of which zidovudine (ZDV)-related reversible anemia is the most com- mon, have shown mild clinical significance.4 – 8 After the PACTG 076 study,9 the 6-week neonatal oral ZDV chemoprophylaxis regimen became standard care. Since 2005, HIV-exposed neonates in the United Kingdom have been treated for 4 weeks,3 in accordance with postexposure prophylaxis (PEP) guidelines in other situations.10 We investigated whether the new 4-week neonatal ZDV regimen has led to a decrease in hematologic toxicity in a large cohort of HIV-uninfected infants. MATERIALS AND METHODS A single-center prospective observational study was con- ducted in a cohort of mother-infant pairs, followed up prospec- tively from January 2000 in a tertiary-care pediatric hospital in Barcelona (Spain). Informed consent was obtained from all women at enrollment and the study protocol was approved by the local Ethics Committee. As per protocol, demographic, clinical, and laboratory data are routinely collected on all patients, and a complete clinical examination and blood tests were performed at every visit (at 2, 3, and 6 weeks, and at 3, 6, and 12 months of age). Laboratory tests included a full blood picture and a viral load assay (proviral HIV-DNA, Amplicor HIV until year 2003, and HIV- RNA quantification, CA HIV Monitor, thereafter; Roche, Basel, Switzerland). HIV-exposed uninfected infants were eligible if they had been exposed to ARV during gestation and had received ZDV monotherapy during the neonatal period. According to current guidelines,1 an infant was defined as HIV-uninfected if 2 or more viral load tests were negative, with one test at age 1 month or older and one test at age 4 months or older. The following exclusion criteria were used: hepatitis C virus (HCV) infection, gestational age at birth less than 36 weeks, and the presence of any other medical condition capable of causing hematologic disorders. Patients were initially defined according to the length of their neonatal ZDV treatment period, a 6-week regimen or a 4-week regimen, according to current guidelines at the time of birth. A univariate assessment of differences in toxicity between the 2 groups Lahoz et al The Pediatric Infectious Disease Journal • Volume 29, Number 4, April 2010 © 2010 Lippincott Williams & Wilkins376 | www.pidj.com
- 14. was conducted usingthe 2 test for categorical variables and the t test for continuous variables. Multivariate regression analyses controlling for several factors known to be associated with hematologic variables (maternal age, ethnicity, drug use, CD4 cell count and plasma viral load, type and length of ARV regimens during pregnancy, and the infant’s sex, gestational age, and weight at birth) were used to assess the association between the different ARV treatment regimens and the outcome variables. All tests were 2-tailed, and a P value Ͻ0.05 was considered significant. Statistical analysis was performed with the SPSS 12.0 Program. RESULTS As of June 2008, 221 infants had been enrolled in the cohort. Among these, 50 patients were excluded because of: neonatal ARV prophylaxis other than ZDV monotherapy (n ϭ 13), HIV infection (n ϭ 5, all of them diagnosed during the first week of life), hepatitis C virus infection (n ϭ 11), gestational age at birth less than 36 weeks (n ϭ 10), and other medical conditions (n ϭ 11). The final study cohort consisted of 171 patients (80 women, 46.8%). Overall, 138 patients (80.7%) received a 6-week neonatal regimen of oral ZDV, while 33 (19.3%) received the new 4-week regimen. Most of the children in the latter group were born from 2005 onwards. All children were exclusively bottle-fed and none received prophylactic trimethoprim-sulfamethoxazole. Data regarding gestation, birth, and neonatal clinical vari- ables are summarized in the Table, Supplemental Digital Content 1, http://links.lww.com/INF/A334. At delivery, mothers in the 6-week ZDV group were significantly younger (P ϭ 0.022), and had been treated more often with stavudine (34% vs. 12%, P ϭ 0.015) and nelfinavir (36% vs. 12%, P ϭ 0.008) and less often with emtricitabine (0% vs. 12%, P ϭ 0.001), abacavir (4% vs. 15%, P ϭ 0.039), and tenofovir (1% vs. 12%, P ϭ 0.014) as part of their highly active antiretroviral therapy (HAART) regimens. Lower mean hemoglobin values (12.1 vs. 13.1 g/dL; P ϭ 0.006) and a higher rate on all Division of Acquired Immunode- ficiency Syndrome toxicity grades11 in hemoglobin concentrations (76% vs. 48%; P ϭ 0.005) at the age of 2 to 3 weeks were observed in infants whose mothers had received ZDV as part of their HAART regimens during pregnancy. These findings persisted in multivariate analyses (odds ratio: 4.28 for any toxicity in hemoglobin values when ZDV was included in maternal therapy; 95% confidence interval ͓CI͔: 1.01–18.05; P ϭ 0.048). No other differences in baseline characteristics or associations between those and hematologic findings were observed (data not shown). Overall, no statistically significant differences were ob- served in hemoglobin, mean corpuscular volume (MCV), neutro- phil, lymphocyte, and platelet counts (Figs. 1A–E) between the 2 groups, except for MCV values at 6 weeks and 3 months of age, which were lower in the 4-week ZDV group (P Ͻ 0.0001 and P ϭ 0.002, respectively; Fig. 1B). In both groups, mean MCV was higher than reference values up to the age of 6 weeks and later became normal. Changes over time in significant abnormalities 6 7 8 9 10 11 12 13 14 15 3 w 6 w 3 m 6 m 12 m 50 60 70 80 90 100 110 120 3w 6w 3m 6m 12m p <.0001 p =.002 0 1000 2000 3000 4000 5000 6000 7000 3w 6w 3m 6m 12m 2000 3000 4000 5000 6000 7000 8000 9000 10000 11000 12000 3w 6w 3m 6m 12m 0 100000 200000 300000 400000 500000 600000 700000 3w 6w 3m 6m 12m hemoglobin (g/dl) mean corpuscular volume (fl) neutrophils per mm3 lymphocytes per mm3 platelets per mm 3 A B C D E FIGURE 1. A to E, Changes over time of the different hematologic parameters according to the length of the neonatal zidovudine regimen, 6 weeks (solid line) or 4 weeks (dotted line). The Pediatric Infectious Disease Journal • Volume 29, Number 4, April 2010 Neonatal Zidovudine Toxicity © 2010 Lippincott Williams & Wilkins www.pidj.com | 377
- 15. (according to theDivision of Acquired Immunodeficiency Syn- drome pediatric grading system11 ) of hemoglobin values and neutrophil counts were similar irrespective of the length of the neonatal ZDV regimen. Normal lymphocyte and platelet counts at all ages were observed for all infants in the study. In the multi- variate assessment, differences in MCV at 6 weeks (P Ͻ 0.0001) and 3 months (P ϭ 0.013) of life were confirmed, and no other significant results were found. Hematologic toxicity did not lead to clinical symptoms in any case; no blood transfusions or treatment discontinuation were required. DISCUSSION Currently, most HIV-infected pregnant women receive HAART. When feasible, use of ZDV as a component of the maternal HAART is recommended, as is the intrapartum intrave- nous ZDV regimen. ZDV also remains the drug of choice in postpartum neonatal chemoprophylaxis.1–3 The 4-week neonatal chemoprophylaxis regimen was first recommended in 2005 by the British guidelines3 and has recently been implemented in Spain.2 The optimal duration of neonatal ZDV chemoprophylaxis has not been appropriately established. A recent clinical trial in Thailand on the prevention on MTCT that used maternal ZDV starting at 28 weeks of pregnancy, plus a loading dose of ZDV during labor, single-dose nevirapine in the mother and the infant, and a 7-day neonatal ZDV treatment achieved a 2% rate of HIV MTCT.12 Although not specifically designed to answer this question, these results suggest that shorter neonatal regimens are as effective as the 6-week regimen in the PACTG 076 TRIAL. Large cohort studies in Europe and the United States indi- cate that perinatal exposure to ARV drugs is associated with minor and mostly clinically insignificant decreases in several hemato- logic parameters, including hemoglobin concentrations and platelet, lymphocyte, CD4 and CD8 cell, and neutrophil counts.4–6,13,14 Ex- cept for hemoglobin, these differences may persist through the age of 18 to 24 months,4–6 or even longer for neutrophil13 and lymphocyte counts.14 In general, the use of HAART during pregnancy and a longer duration of exposure lead to larger decreases.4,7,14 The observational nature of our study, the small sample size, the lack of a control group of ARV-unexposed infants, and the fact that, overall, 93% of the mothers received HAART during pregnancy did not allow us to confirm these findings. Nonetheless, the absence of differences in most hematologic indices when the length of the neonatal ZDV regimen was taken into account suggests that this toxicity is mainly due to the use of ARV during pregnancy. The use of historic controls instead of concurrent ones could represent a potential for bias and limit the validity of the study; however, there were no dissimilarities in the baseline characteristics, the endpoints were objective and adequate data were available to allow for the comparison between both regimen groups. Short-term toxicity of infant ZDV prophylaxis has been minimal, consisting primarily of transient macrocytic anemia. Several studies suggest that hematologic toxicity increases when intravenous ZDV5 or combined neonatal ZDV/lamivudine prophy- laxis5,15 are used, with increasing numbers of patients requiring treatment discontinuation or blood transfusions. The change in hemoglobin value in our study, with a nadir occurring at 6 weeks of age, is similar to that in the PACTG 076 trial.9 In contrast with other hematologic parameters, hemoglobin concentrations tend to normalize at 2 to 3 months of age, early after the exposure to any ARV finishes.4 –9 The in vitro inhibitory effect of ZDV on hema- topoietic progenitors was reported early on by Dainiak et al,16 and it is more severe on the fetal and neonatal erythroid lineages.17 Although the lower hemoglobin levels at age 2 to 3 weeks we describe were only associated with the maternal use of ZDV during gestation, the significant differences in MCV values be- tween groups at 6 and at 12 weeks of age suggest that the 4-week regimen allows an earlier recovery of the erythroid cell lineage in bone marrow. In conclusion, our results suggest that the use of HAART during pregnancy, especially if this regimen includes ZDV, is critical for the development of neonatal anemia; moreover, the length of the ZDV neonatal regimen likely represents an additive toxic effect. Nowadays, most HIV-infected pregnant women re- ceive HAART. Current guidelines on the prevention of HIV MTCT allow a vaginal delivery when maternal viral load near the time of delivery is below 1000 HIV-RNA copies/mL and recom- mend an elective cesarean section when maternal viral load near the time of delivery is greater than 1000 HIV-RNA copies/mL,1 hence minimizing the risk for the newborn to come into contact with contaminated blood and the role of PEP. Likewise, HIV infection may be presumptively excluded by 1 month of age, before the completion of the 6-week PEP.1 Present PEP guidelines in other situations recommend 4-week regimens of different com- bined ARV.10 Whether the neonatal 4-week oral ZDV regimen, or even shorter ones, are as effective as the 6-week regimen in preventing HIV MTCT can only be confirmed by means of a clinical trial. Until then, observational studies with close monitor- ing of ZDV-exposed infants and assessment of their clinical data contribute to the body of evidence on the effectiveness and safety of the 4-week infant PEP, and provide support for this new recommendation. REFERENCES 1. Centers for Disease Control and Prevention. Public Health Service Task Force. Perinatal HIV Guidelines Working Group. Recommendations for use of antiretroviral drugs in pregnant HIV-1-infected women for maternal health and interventions to reduce perinatal HIV-1 transmission in the United States. July 2008. Available at: http://AIDSinfo.nih.gov. Accessed September 1, 2009. 2. Recomendaciones de la Secretaria del Plan Nacional sobre el Sida, el Grupo de Estudio de Sida, la Sociedad Espan˜ola de Ginecología y Obstetrícia y la Asociacio´n Espan˜ola de Pediatría para el seguimiento de la infeccio´n por el VIH con relacio´n a la reproduccio´n, el embarazo y la prevencio´n de la transmisio´n vertical. Available at: www.msc.es/ciudadanos/enfLesiones/ enfTransmisibles/sida/publicaciones/publicaciones.htm. Accessed Septem- ber 1, 2009. 3. Ruiter A, Mercey D, Anderson J, et al. British HIV association and children’s HIV association guidelines for the management of HIV infection in pregnant women 2008. HIV Med. 2008;9:452–502. 4. Le Chenadec J, Mayaux MJ, Guihenneuc-Jouyaux C, et al. Perinatal antiretroviral treatment and hematopoiesis in HIV-uninfected infants. AIDS. 2003;17:2053–2061. 5. Bunders M, Bekker V, Scherpbier HJ, et al. Haematological parameters of HIV-1-uninfected infants born to HIV-1 infected mothers. Acta Paediatr. 2005;94:1571–1577. 6. Pacheco SE, McIntosh K, Lu M, et al. Effect of perinatal antiretroviral drug exposure on hematologic values in HIV-uninfected children: an analysis of the women and infants transmission study. J Infect Dis. 2006;194:1089– 1097. 7. Feitena-Sperling C, Weizsaecker K, Bu¨hrer C, et al. Hematologic effects of maternal antiretroviral therapy and transmission prophylaxis in HIV-1- exposed uninfected newborn infants. J Acquir Immune Defic Syndr. 2007; 45:43–51. 8. Mussi-Pinhata MM, Rego MA, Freimanis L, et al. Maternal antiretrovirals and hepatic enzyme, hematologic abnormalities among human immunode- ficiency virus type 1-uninfected infants. Pediatr Infect Dis J. 2007;26: 1032–1037. 9. Connor EM, Sperling RS, Gelber R, et al; Pediatric AIDS Clinical Trials Group Protocol 076 Study Group. Reduction of maternal-infant transmis- sion of human immunodeficiency virus type 1 with zidovudine treatment. N Engl J Med. 1994;331:1173–1180. Lahoz et al The Pediatric Infectious Disease Journal • Volume 29, Number 4, April 2010 © 2010 Lippincott Williams & Wilkins378 | www.pidj.com
- 16. 10. Centers forDisease Control and Prevention. Updated US Public Health Service guidelines for the management of occupational exposures to HIV and recommendations for postexposure prophylaxis. MMWR Recomm Rep. 2005;54(RR-9):8. 11. Division of AIDS table for grading the severity of adult and pediatric adverse events. December 2004. (niaid.nih.gov website). Available at: http://www3.niaid.nig.gov/research/resources/DAIDSClinRsrch/PDF/Safe- ty/DAIDSAEGradingTable.pdf. Accessed September 1, 2009. 12. Lallemant M, Jourdain G, Le Coeur S, et al. Single-dose perinatal nevirap- ine plus standard zidovudine to prevent mother-to-child transmission of HIV-1 in Thailand. N Engl J Med. 2004;351:217–228. 13. European Collaborative Study. Levels and patterns of neutrophil cell counts over the first 8 years of life in children of HIV-1-infected mothers. AIDS. 2004;18:2009–2017. 14. Bunders M, Thorne C, Newell ML; European Collaborative Study. Mater- nal and infant factors and lymphocyte, CD4 and CD8 cell counts in children of HIV-1-infected mothers. AIDS. 2005;19:1071–1079. 15. Mandelbrot L, Landreau-Mascaro A, Rekacewicz C, et al. Lamivudine-zidovudine combination for prevention of maternal-infant transmission of HIV-1. JAMA. 2001;285:2083–2093. 16. Dainiak N, Worthington M, Riordan MA, et al. 3Ј-Azido-3Ј-deoxythymidine (AZT) inhibits proliferation in vitro of human haematopoietic progenitor cells. Br J Haematol. 1988;69:299–304. 17. Shah MM, Li Y, Christensen RD. Effects of perinatal zidovudine on hemato- poiesis: a comparison of effects on progenitors from human fetuses versus mothers. AIDS. 1996;10:1239–1247. MULTIDRUG-RESISTANT ENTEROCOCCUS FAECIUM MENINGITIS IN A TODDLER CHARACTERIZATION OF THE ORGANISM AND SUCCESSFUL TREATMENT WITH INTRAVENTRICULAR DAPTOMYCIN AND INTRAVENOUS TIGECYCLINE Heather B. Jaspan, MD, PhD,*† Adam W. Brothers, PharmD,* Angela J. P. Campbell, MD,*†‡ John K. McGuire, MD,*† Samuel R. Browd, MD, PhD,§ Thomas J. Manley, MD,*‡ Daniel Pak, PharmD,† and Scott J. Weissman, MD*† Abstract: A case of enterococcal meningitis in a toddler is presented. The organism was highly resistant to all drugs previously used for pediatric Gram-positive meningitis. She was successfully treated with intraventric- ular and intravenous daptomycin and intravenous tigecycline. The organ- ism was characterized as a member of CC17, a notorious emerging nosocomial clone of Enterococcus faecium. Key Words: vancomycin-resistant Enterococcus, tigecycline, daptomycin, meningitis, children Accepted for publication October 27, 2009. From the *Department of Pediatrics, Seattle Children’s Hospital, Seattle WA; †Department of Pediatrics, University of Washington, Seattle, WA; ‡Fred Hutchinson Cancer Research Institute, Seattle WA; and §Department of Neurological Surgery, Seattle Children’s Hospital, Seattle WA. H.B.J. is supported in part by NIH Training grant 5T32HD007233-28. Address for correspondence: Heather Jaspan, MD, PhD, Seattle Children’s Hospital, 4800 Sandpoint Way NE, Seattle, WA 98105. E-mail: heather.jaspan@sbri.org. DOI: 10.1097/INF.0b013e3181c806d8 A 21-month-old girl received an HLA-matched, haploidentical peripheral blood stem cell transplant with natural killer cell add-back for treatment of refractory acute bilineage leukemia. She received intense myelosuppression with a total of 14 daily doses of OKT3 (muromonab). On the third post-transplant day, she developed fever. Peripheral blood culture obtained via central venous catheter (CVC) had growth of Enterococcus faecium resistant to vancomycin (Table 1, isolate 1). Vancomycin-resistant enterococci (VRE) had been recovered from her stool on routine surveillance one month earlier. Linezolid was administered intravenously in a dosage of 10 mg/kg every 8 hours, but fever persisted and blood cultures showed growth of E. faecium, even after removal of the CVC 4 days after starting antimicrobial therapy. Daptomycin (4 mg/kg every 12 hours) was added to the regimen 6 days after linezolid therapy was initiated. Antibiotic susceptibility testing of the organism was repeated and had not changed since Day 0 (Table 1, isolate 2). When peripheral blood cultures demonstrated continued growth of VRE, an echocardiogram was obtained, ten days after start of antimicrobial therapy. The study identified echogenic linear foci in the innominate vein, likely representing sheath thrombi from the previously removed CVC. An intravenous heparin drip was initiated to prevent extension of the thrombus. After 20 days of therapy with Linezolid, 14 days with daptomycin, and 7 days with heparin, blood cultures were sterilized, with the last positive blood culture having been obtained 20 days after the first (Table 1, isolate 3). A repeat echocardiogram showed the innominate vein thrombi to be smaller. Despite sterile blood cultures, the patient continued to have fevers and developed emesis. Eight days after the last positive blood culture, she developed acute mental status changes. When computed tomography of the head showed acute hydrocephalus, an external ventriculostomy drain (EVD) was emergently placed. Microscopic examination of cerebrospinal fluid (CSF) revealed Gram-positive cocci, and culture subsequently demonstrated growth of E. faecium (Table 1, isolate 4). When antimicrobial susceptibility testing revealed that the CSF isolate was resistant to Linezolid, this drug was discon- tinued. Other antibiotic options were limited. Because of the poor pene- tration of systemic daptomycin into the CSF,1 the risks and benefits of direct intraventricular daptomycin therapy via ventriculostomy were dis- cussed with the family, who agreed to proceed with this therapy. On the basis of a previous report of intraventricular daptomy- cin use in an adult,2 daptomycin 2.5 mg in 5 mL normal saline was administered via ventriculostomy tubing every 24 hours, locked for 30 minutes, and then reopened to continue CSF drainage. To reduce the risk of chemical meningitis due to direct instillation of the antimicro- bial, we initiated intravenous dexamethasone, 1 mg every 6 hours for 5 days, on day 2 of intraventricular daptomycin. No clinical signs of increased central nervous system irritation (such as increased emesis or irritability) were observed, nor were increases in CSF pleocytosis or protein noted on daily monitoring. Peak and trough concentrations of daptomycin measured in CSF specimens from the EVD at the end of the intraventricular infusion and 30 minutes before the next dose were 24.44 and 2.97 mg/L, respectively. The measurements were obtained at the Center for Anti-Infective Research and Development (Hartford, CT), using high-performance liquid chromatography (HPLC). TABLE 1. Evolution of the Susceptibilities of the Enterococcus Isolates From Blood and CSF Drug Isolate 1 Blood Day 0 MIC Isolate 2 Blood Day 7 MIC Isolate 3 Blood Day 15 MIC Isolate 4 CSF Day 25 MIC Vancomycin Ͼ256 (R) Ͼ256 (R) Ͼ256 (R) Ͼ256 (R) Chloramphenicol* S I I R Doxycycline* S S S R Linezolid* S S S R Synercid* S S S R Rifampin* R R R R Daptomycin 1 (S) NT NT 2 (S) Tigecycline NT 0.25 (NI) 0.05 (NI) 0.125 (NI) * Susceptibility testing for these antimicrobials were performed using disk diffusion method and therefore MICs are not available. MIC indicates minimum inhibitory concentration; S, susceptible; I, intermediate; R, resistant; NT, not tested; CSF, cerebral spinal fluid; NI, no interpretation available. The Pediatric Infectious Disease Journal • Volume 29, Number 4, April 2010 Enterococcal Meningitis © 2010 Lippincott Williams & Wilkins www.pidj.com | 379
- 17. Gram stain andculture of the CSF remained positive during the first 3 days of intraventricular therapy. The only remaining agent to which the isolate was susceptible was tigecycline. Based on standard adult dosing and a report of a mean site-to-serum area under the concentration-time curve (AUC) ratio of 0.11 for tige- cycline in CSF of patients with noninflamed meninges,3 a loading dose of 3 mg/kg i.v. was extrapolated, with maintenance dosing of 2 mg/kg i.v. every 8 hours to maximize CSF penetration. Two days after tigecycline was added, the patient’s CSF sterilized. Platelet and white blood cell counts were monitored daily and remained at baseline. Mildly elevated hepatic transaminases developed on treatment day 8 of tigecycline, but the patient was receiving concomitant hepatotoxic drugs. Serum tigecycline concentrations were measured by HPLC at 0.75, 4, and 7 hours after the half-hour drug infusion, and an AUC of 8.7 mg h/L for an 8-hour dosing interval was calculated using the trapezoidal method. Tigecycline concentrations in CSF collected from the EVD at 0.75 and 4 hours postinfusion were below the detectable level of 0.05 mg/L. Intraventicular daptomycin was continued for 7 days after the date of the first negative CSF culture, although the dosage was decreased to 2 mg every 24 hours for 2 days before stopping, to minimize the risk of toxicity. Tigecycline was given for a total of 14 days. A ventriculoperitoneal shunt was placed after 10 days of sterile CSF cultures. Parenteral daptomycin was continued for 6 weeks after the first sterile blood culture due to the thrombotic nidus of infection. The patient returned to her baseline neurologic functioning. To determine the phylogenetic background and confirm the clonality of the VRE isolates, multilocus sequence typing was per- formed as previously described.4 According to the publicly available multilocus sequence typing database (http://efaecium.mlst.net/), all of the tested clinical isolates belonged to ST412, which is a member of the epidemic clonal complex 17 (CC17). CC17 is now widely recog- nized as a hospital-adapted E. faecium subpopulation,5 and member strains typically carry multiple virulence-associated traits. DISCUSSION We believe that this is the first reported case of pediatric meningitis successfully treated with intraventricular daptomycin. It is also the first report of tigecycline use in a toddler. While use of these agents in combination precludes crediting one or the other with the predominant curative role, both were well tolerated, and the patient’s CSF was successfully sterilized of this highly-resistant isolate, with no evidence of recurrence at 3 months of follow-up. Vancomycin-resistant E. faecium is an emerging pathogen for hospitalized and immunocompromised patients. Most enterococci have intrinsic resistance to multiple classes of antimicrobial agents, including cephalosporins and semisynthetic penicillinase-resistant penicillins (eg, oxacillin), as well as to clinically achievable serum concentrations of aminoglycosides. The toxicity of chloramphenicol has proscribed its use in most industrialized countries. Therefore, until recently, antimicrobial therapy for the treatment of VRE was limited. Although more options have become available with the approval of novel antimicrobial agents, there is usually a delay in approval of such agents for use in children. Linezolid was approved by the Food and Drug Administration for use in children in 2002,6 but as of this writing, daptomycin has not been so approved, although a clinical trial is underway (available at: http://clinicaltrials.gov/ct2/show/ NCT00136292). Tigecycline is a treatment option for adults with serious infections caused by methicillin-resistant Staphylococcus aureus (MRSA) or VRE. Pharmacokinetic data from the manu- facturer indicate that a dose of 1 mg/kg every 12 hours in pediatric patients 8 years of age and older provides an AUC that is comparable to that for standard adult dosing (Wyeth Laboratory). No data are available on the safety of this drug in pediatrics, as use of the related agent tetracycline in children less than 8 years of age has the potential risk of tooth discoloration. Daptomycin has been used for children with MRSA and VRE infections in situations where few alternatives exist. In a retrospective review of children with VRE or MRSA infections (median age, 6.5 years), doses of 4 to 6 mg/kg of dapto- mycin were used as adjunct therapy with successful outcomes.7 Options for the treatment of E. faecium meningitis in children are further limited, as many of the available drugs do not reach therapeutic CSF concentrations. Linezolid achieves excellent CSF penetration, with a CSF-to-serum ratio of 0.92.8 Linezolid has been used successfully for central nervous system infections with E. fae- cium in children.9 The organism isolated from the CSF in this case was resistant to Linezolid. Daptomycin has not been studied for meningitis in humans. In experimental animal meningitis models, CSF penetration is 5% with inflamed meninges.1 It has been used intraventricularly in one reported case in an adult.2 At that patient’s l dose of 5 mg every 3 days, peak and trough levels from the CSF were 13.9 and 9.9 mg/L. Sterile CSF and survival of infection were achieved. Our patient’s CSF drug concentrations were substantially lower, yet still achieved a good outcome. Although CSF tigecycline was 11% of the serum concentration in healthy adult patients receiving a 100 mg dose,3 in our case, the intraventricular concentration of tigecycline was below the limit of detection. It is unclear why this was the case given that peripheral blood concentrations were substantial. There are no studies assessing the impact of meningeal inflammation on CSF levels of the drug, but since we obtained the measured CSF sample several days after completion of dexamethasone therapy (and when CSF cultures were sterile), there was unlikely to have been any inflammation of the meninges which could have affected CSF penetration. The AUC calculated from our patient’s serum drug concentrations was higher than the mean AUC in the adult study,3 and what is reported in the drug’s package insert for 24 hours after a single loading dose of 100 mg.10 Nonetheless, doses of 1 mg/kg every 12 hours would be sufficient for patients less than 8 years old in a situation where no other options exist, although it is unclear whether the tigecycline had any therapeutic benefit for our patient’s meningitis. Decrease in enterococcal susceptibility to Linezolid after ex- posure to this drug has previously been reported11 to be the result of a single gene mutation. In this report, resistance to quinupristin/ dalfopristin, chloramphenicol, and doxycycline also emerged, despite lack of exposure to these agents. CC17, the group to which our isolate belongs, is a notorious emerging hospital-acquired pathogen, with multiple inducible chromosomal and plasmid-located resistance and virulence genes.5 This case emphasizes the urgent need for pediatric clinical trials of antibiotics for emerging resistant organisms. It also provides clinical guidance in the use of i.v. tigecycline or intraventricular daptomycin in children where there are no better alternatives. ACKNOWLEDGMENTS The authors thank all of the health care providers who were involved in the care of our patient, including Cathy Cornell, Joan Suver, Dr. Laurie Burroughs, Dr. Farah Cassis-Ghavami, Dr. Jane Burns, and David Nicolau. Additionally, the authors thank Xuan Qin for her expert microbiological advice, Dr. Ann Woolfrey for editorial input, and Christina Sutherland at Hartford Hospital who performed the HPLC for serum drug concentrations. REFERENCES 1. Gerber P, Stucki A, Acosta F, et al. Daptomycin is more efficacious than vancomycin against a methicillin-susceptible Staphylococcus aureus in experimental meningitis. J Antimicrob Chemother. 2006;57:720–723. Jaspan et al The Pediatric Infectious Disease Journal • Volume 29, Number 4, April 2010 © 2010 Lippincott Williams & Wilkins380 | www.pidj.com
- 18. 2. Elvy J,Porter T, Brown E. Treatment of external drain-associated ventric- ulitis caused by Enterococcus faecalis with intraventricular daptomycin. J Antimicrob Chemother. 2008;61:461–462. 3. Rodvold KA, Gotfried MH, Cwik M, et al. Serum, tissue and body fluid concentrations of tigecycline after a single 100 mg dose. J Antimicrob Chemother. 2006;58:1221–1229. 4. Homan WL, Tribe D, Poznanski S, et al. Multilocus sequence typing scheme for Enterococcus faecium. J Clin Microbiol. 2002;40:1963–1971. 5. Top J, Willems R, Bonten M. Emergence of CC17 Enterococcus faecium: from commensal to hospital-adapted pathogen. FEMS Immunol Med Mi- crobiol. 2008;52:297–308. 6. Food and Drug Administration Advisory Committee. Presentation: Zyvox (linezolid). Available at: http://www.fda.gov/ohrms/dockets/ac/06/slides/ 2006- 4254s_02_Shapiro_Linezolid.ppt. Accessed June 29, 2008. 7. Ardura MI, Mejıas A, Katz K, et al. Daptomycin for invasive Gram-positive bacterial infections in children. Pediatr Infect Dis. 2007;26:1128–1132. 8. Krueger WA, Kotler B, Will BE, et al. Treatment of meningitis due to MRSE with linezolid. J Clin Microbiol. 2004;42:929–932. 9. da Silva PS, Monteiro Neto H, Sejas LM. Successful treatment of vanco- mycin-resistant Enterococcus ventriculitis in a child. Braz J Infect Dis. 2007;11:297–299. 10. Tygacil (tigecycline) ͓package insert͔. Philadelphia, PA: Wyeth Pharma- ceuticals; June 2007. 11. Raad II, Hanna HA, Hachem RY, et al. Clinical-use-associated decrease in susceptibility of vancomycin resistant Enterococcus faecium to linezolid: a comparison with quinupristin-dalfopristin. Antimicrob Agents Chemother. 2004;48:3583–3585. LATE-ONSET SEPSIS IN A PRETERM TWIN MAY HARBINGER LIFE-THREATENING SEPSIS FOR THE ASYMPTOMATIC CO-TWIN Jayaram R. Pai, MRCPCH,* Catherine H. Tremlett, MD, MSc, FRCPath,† and Paul Clarke, MD, FRCPCH, MRCP(UK)* Abstract: We report extremely preterm twins who developed late-onset Escherichia coli sepsis in the second postnatal week within a short time of each other. The asymptomatic twin was not treated initially, and within 2 days developed life-threatening septicemia and meningitis, followed by other serious morbidity. Occurrence of late-onset sepsis in a twin should prompt concurrent investigation and consideration of presumptive treat- ment of the apparently asymptomatic co-twin. Key Words: twins, infection, prophylaxis, coliform, Escherichia coli Accepted for publication October 2, 2009. From the *Neonatal Intensive Care Unit, Norfolk and Norwich University Hospitals NHS Foundation Trust, Norwich, United Kingdom; and †Depart- ment of Medical Microbiology, Norfolk and Norwich University Hospitals NHS Foundation Trust, Norwich, United Kingdom. Address for correspondence: Paul Clarke, MD, Neonatal Intensive Care Unit, Norfolk and Norwich University Hospitals NHS Foundation Trust, Norwich, NR4 7UY United Kingdom. E-mail: paul.clarke@ nnuh.nhs.uk. DOI: 10.1097/INF.0b013e3181c42545 Late-onset sepsis is a life-threatening complication affecting Ͼ20% of very low birth weight (VLBW; Ͻ1500 g) infants.1 In developed countries, coliforms are the third commonest organisms (after coagulase-negative staphylococci and Staphylococcus au- reus) responsible for late-onset sepsis in VLBW infants in the neonatal intensive care unit (NICU), and are among the most lethal of infecting bacterial pathogens.1 One study showed the incidence of late-onset Escherichia coli sepsis in VLBW infants to have doubled in recent years, from ϳ10 cases per 1000 VLBW admis- sions in 1979–1992, to ϳ22 cases per 1000 VLBW admissions in 1997–2006.2 An increased incidence of group B streptococcal co-sepsis has been reported in twins,3,4 providing a sound basis for presump- tive treatment of both twins if one presents with early-onset sepsis. There has been no prior report to suggest an increased risk of late-onset coliform co-sepsis in twins, and no clear evidence to mandate invasive testing and prophylactic treatment of an asymp- tomatic co-twin when one twin presents with late-onset sepsis. We report the case of extremely preterm twins both of whom developed coliform sepsis in the second postnatal week. The un- treated, initially-asymptomatic co-twin developed life-threatening E. coli infection within 2 days of the index twin becoming septic. CASE REPORT Dichorionic diamniotic twins were delivered vaginally at 27 weeks and 3 days gestation following spontaneous onset of labor. Mother, 23-year-old, had an episode of cystitis during pregnancy, and membranes had ruptured spontaneously 33 hours before de- livery. She had received 2 doses of betamethasone antenatally. Both twins were in good condition at delivery and were electively intubated, given surfactant, and admitted to our NICU for ongoing ventilation. Twin 1 was a female and weighed 1030 g; twin 2 was a male and weighed 1149 g. Both were extubated to nasal contin- uous positive airways pressure within 24 hours of delivery. In view of the risk factors for sepsis, each infant had a blood culture, complete blood count (CBC), and C-reactive protein (CRP) sent on admission, and was treated with intravenous benzylpenicillin and gentamicin for 5 days. Blood cultures returned negative at 48 hours in both cases. Twin 2. On postnatal day 10, twin 2 became unwell with overt clinical signs of sepsis. He had frequent drops in oxygen satura- tions, poor tone, and reduced activity. He was intubated, venti- lated, and underwent CBC, CRP, blood and urine cultures, chest radiography, and cerebrospinal fluid (CSF) studies. The percuta- neous central venous catheter being used for parenteral nutrition was concurrently removed as a potential source of infection. Blood results showed CRP 33 mg/L, white blood cells 17.2 ϫ 109 /L, and neutrophils 12.2 ϫ 109 /L. Treatment was commenced with van- comycin and gentamicin. His CSF was blood-stained but analysis was otherwise normal (white blood cells 42 ϫ 106 /L, red blood cells 43200 ϫ 106 /L, no organisms seen), and culture was sterile. The blood culture taken on day 10 grew Gram-negative rods on day 12, at which point vancomycin was stopped and replaced with ceftazidime. On day 13 the definitive blood culture result was confirmed as a growth of E. coli, susceptible to cephalosporins and gentamicin but resistant to amoxicillin. Ceftazidime was stopped and cefotaxime substituted. Gentamicin was continued for 7 days and cefotaxime for a total of 10 days. Urine culture showed no growth. Cranial ultrasound scans were normal. Twin 1. On day 10, at the time when twin 2 had presented with sepsis, twin 1 was clinically stable and her CRP was Ͻ3 mg/L. Laboratory tests for infection were not done and antibiotics were not given. Instead, close clinical observation and a low threshold for laboratory assessment of infection and antibiotic treatment were advised. Thirty-four hours later (postnatal day 12), she became acutely unwell with an unstable temperature, increased breathing effort, tachycardia, and lactic acidosis. She was reintu- bated, ventilated, and underwent CBC, blood and urine cultures, chest radiography, and lumbar puncture. CSF examination indi- cated bacterial meningitis (white cell count 3180 ϫ 106 /L, 90% neutrophils, protein 5.3 g/L, glucose 1.6 mmol/L, and Gram- negative organisms were visible on the Gram stain). Concomitant blood glucose was 7.5 mmol/L. Treatment was commenced with meropenem in a dosage of 40 mg/kg every 8 hours. Blood and CSF cultures subsequently confirmed E. coli bacteremia and meningitis. The Pediatric Infectious Disease Journal • Volume 29, Number 4, April 2010 Coliform Sepsis in Preterm Twins © 2010 Lippincott Williams & Wilkins www.pidj.com | 381
- 19. The E. colihad an identical antibiotic susceptibility pattern as that from twin 2, and subsequent typing by the Health Protection Agency Centre for Infections (London, United Kingdom) showed the strains from both twins to be E. coli serotype 031 K1. Review of the result of a throat swab of twin 1 that had been taken on admission to the NICU showed a coliform growth with an identical antibiotic susceptibility pattern as the E. coli serotype 031 K1. This coliform was not typed or identified further. Twin 1 was critically ill for many days: she had pulmonary hemorrhage and respiratory distress that required high-frequency oscillatory ventilation for 1 week and positive pressure ventilation for a further 17 days, and hypotension that required inotropic support for 2 weeks. She developed impaired renal function and thrombocytopenia (nadir platelet count 15 ϫ 109 /L) and needed platelet transfusions. She suffered ischemic injury to her right index finger, and on day 19 developed necrotizing enterocolitis with intestinal perforation. She underwent laparotomy and ileos- tomy formation on day 22. She had repeated lumbar punctures on days 26 and 31, and both these CSF samples were sterile. Meropenem was changed to cefotaxime and vancomycin on day 22, and she received both these antibiotics for 24 days in total; metronidazole was also given for 14 days following the onset of necrotizing enterocolitis. Subse- quent cranial ultrasound scans showed only a small subependymal hemorrhage and no evidence of parenchymal brain injury. Both twins were eventually discharged home. Follow-up examination at the age of 12 months (corrected for prematurity: 9 months) showed mild gross motor developmental delay in both infants. DISCUSSION These extremely preterm twins developed life-threatening E. coli septicemia in the second week of life and within a short time of each other. The asymptomatic co-twin was reviewed but not treated at the time when the index twin developed overt infection. Despite appearing clinically well, the co-twin presum- ably had asymptomatic bacteremia at this time because just 34 hours later she had developed serious infection, including menin- gitis. While twins per se appear at no greater risk of invasive infection than singletons,5 it is recognized for group B streptococ- cal infection that being the co-twin of an infected twin sibling does greatly increase the risk of sepsis.3–5 We believe that this is the first report to suggest that being a co-twin of a twin infected with E. coli may substantially increase the risk of late-onset invasive E. coli sepsis. These twins had a number of risk factors for sepsis, includ- ing extreme prematurity and delivery after prolonged rupture of membranes. Both had received 5 days of antibiotics after birth, but initial blood cultures were sterile. Twin 1 had a throat swab taken on admission, which grew a coliform with an identical antibiotic susceptibility pattern as the invasive E. coli. We speculate that both babies were colonized perinatally with E. coli and that the 5-day long initial antibiotic course did not clear the congenital colonization. Additional risk factors associated with neonatal in- tensive care (eg, mechanical ventilation, nasal continuous positive airways pressure, presence of indwelling central venous catheters, and parenteral nutrition delivery), may have increased the risk of invasive E. coli sepsis in both these infants. Persistent colonization and recurrence of disease has also been shown in neonatal group B streptococcal infection.6,7 This case report provides evidence to support concurrent evaluation and consideration of empiric treatment of an apparently- well preterm co-twin when a twin sibling presents with late-onset sepsis. In the present case, enhanced clinical surveillance for signs of infection clearly proved to be insufficient. Our experience with these twins, both of whom had life-threatening E. coli infection, would support the practice of undertaking laboratory investiga- tions to seek evidence of early infection in a clinically well preterm co-twin, and consideration of presumptive treatment with appro- priate antibiotics. We suggest that studies should include blood and urine cultures, chest radiograph and CSF analysis, and that anti- biotics be given at least until invasive infection is disproven in the asymptomatic twin by negative blood, urine, and CSF cultures. While empiric antibiotic treatment may not guarantee prevention of late-onset sepsis in such circumstances,8 such practice would likely have prevented or aborted the life-threatening E. coli septi- cemia, meningitis, renal failure, and protracted ventilation suffered by this initially-asymptomatic co-twin. REFERENCES 1. Stoll BJ, Hansen N, Fanaroff AA, et al. Late-onset sepsis in very low birth weight neonates: the experience of the NICHD Neonatal Research Network. Pediatrics. 2002;110:285–291. 2. Bizzarro MJ, Dembry LM, Baltimore RS, et al. Changing patterns in neonatal Escherichia coli sepsis and ampicillin resistance in the era of intrapartum antibiotic prophylaxis. Pediatrics. 2008;121:689–696. 3. Edwards MS, Jackson CV, Baker CJ. Increased risk of group B streptococcal disease in twins. JAMA. 1981;245:2044–2046. 4. Benitz WE, Gould JB, Druzin ML. Risk factors for early-onset group B streptococcal sepsis: estimation of odds ratios by critical literature review. Pediatrics. 1999;103:e77. 5. Benitz WE. Re: late-onset group B streptococcal infection in identical twins: insight to disease pathogenesis. J Perinatol. 2002;22:685. 6. Moylett EH, Fernandez M, Rench MA, et al. A 5-year review of recurrent group B streptococcal disease: lessons from twin infants. Clin Infect Dis. 2000;30:282–287. 7. Doran KS, Benoit VM, Gertz RE, et al. Late-onset group B streptococcal infection in identical twins: insight to disease pathogenesis. J Perinatol. 2002;22:326–330. 8. Rubin EE, McDonald JC. Group B streptococcal disease in twins: failure of empiric therapy to prevent late onset disease in the second twin. Pediatr Infect Dis J. 1991;10:621–623. Pai et al The Pediatric Infectious Disease Journal • Volume 29, Number 4, April 2010 © 2010 Lippincott Williams & Wilkins382 | www.pidj.com