ABSTRACT

Objective

To 1) describe the correlation between the zones of inhibition in 1-µg oxacillin disk diffusion (ODD) tests and penicillin and ceftriaxone minimum inhibitory concentrations (MICs) of meningeal and non-meningeal strains of Streptococcus pneumoniae and 2) evaluate the usefulness of the ODD test as a predictor of susceptibility to penicillin in S. pneumoniae and as a quick and cost-effective method easily implemented in a routine clinical laboratory setting.

Methods

S. pneumoniae isolates from healthy nasopharyngeal carriers less than 2 years old, obtained in a multicentric cross-sectional study conducted in various Peruvian hospitals and health centers from 2007 to 2009, were analyzed. Using Clinical and Laboratory Standards Institute (CLSI) breakpoints, the correlation between the zones of inhibition of the ODD test and the MICs of penicillin and ceftriaxone was determined.

Results

Of the 571 S. pneumoniae isolates, 314 (55%) showed resistance to penicillin (MIC ≥ 0.12 µg/mL) and 124 (21.7%) showed resistance to ceftriaxone (MIC ≥ 1 µg/mL). Comparison of the ODD test zones of inhibition and the penicillin MICs, using the CLSI meningeal breakpoints, showed good correlation (Cohen’s kappa coefficient = 0.8239).

Conclusions

There was good correlation between ODD zones of inhibition and penicillin meningeal breakpoints but weak correlation between the ODD results and non-meningeal breakpoints for both penicillin and ceftriaxone. Therefore, the ODD test appears to be a useful tool for predicting penicillin resistance in cases of meningeal strains of S. pneumoniae, particularly in low- and middle- income countries, where MIC determination is not routinely available.

Key words
Streptococcus pneumoniae; oxacillin; Peru; Latin America

RESUMEN

Objetivo

1) Describir la correlación entre las zonas de inhibición observadas en la prueba de difusión con discos de oxacilina de 1 µg y la concentración inhibitoria mínima (CIM) de penicilina y ceftriaxona frente a cepas meníngeas y no meníngeas de Streptococcus pneumoniae y 2) evaluar si la prueba de difusión con discos de oxacilina permite predecir la sensibilidad de S. pneumoniae a la penicilina y sirve como método rápido y eficaz en función de los costos, y resulta fácil de aplicar en los laboratorios clínicos ordinarios.

Métodos

Se analizaron colonias de S. pneumoniae aisladas de la nasofaringe de portadores sanos menores de 2 años obtenidas en un estudio transversal multicéntrico realizado en diversos hospitales y centros de salud del Perú entre los años 2007 y 2009. Se determinó la correlación entre las zonas de inhibición observadas en la prueba de difusión con discos y la CIM de la penicilina y la ceftriaxona utilizando los valores críticos definidos por el Instituto de Estándares Clínicos y de Laboratorio.

Resultados

De las 571 colonias aisladas de S. pneumoniae, 314 (55 %) presentaron resistencia a la penicilina (CIM ≥ 0,12 µg/ml) y 124 (21,7%), resistencia a la ceftriaxona (CIM ≥ 1 µg/ml). Se observó una buena correlación (coeficiente κ de Cohen = 0,8239) entre las zonas de inhibición de la prueba de difusión con discos y la CIM de la penicilina utilizando los valores críticos del Instituto respecto de las cepas meníngeas.

Conclusiones

Se encontró una buena correlación entre las zonas de inhibición de la prueba de difusión con discos y los valores críticos de CIM de la penicilina respecto de las cepas meníngeas, pero una correlación débil entre los resultados de la prueba de difusión y los valores críticos tanto de la penicilina como de la ceftriaxona respecto de las cepas no meníngeas. Por consiguiente, la prueba de difusión con discos es un método de utilidad para predecir la resistencia a la penicilina de las cepas meníngeas de S. pneumoniae, en particular en los países de ingresos bajos y medianos, donde no suele ser posible determinar la CIM.

Palabras clave
Streptococcus pneumoniae; oxacilina; Perú; América Latina

Streptococcus pneumoniae is the most common cause of community-acquired bacterial pneumonia, meningitis, and bacteremia and non-invasive diseases such as sinusitis and otitis media (¹1 Chiou CC, Hseih KS. Pneumococcal infection in children: rational antibiotic choice for drug-resistant Streptococcus pneumoniae. Acta Paediatr Taiwan. 2003;44(2):67–74.). Traditionally, the use of penicillin has been recommended for respiratory tract infections, and the administration of third-generation cephalosporins, such as ceftriaxone or cefotaxime, has been recommended for meningitis treatments (²2 Waisbourd-Zinman O, Bilavsky E, Tirosh N, Samra Z, Amir J. Penicillin and ceftriaxone susceptibility of Streptococcus pneumoniae isolated from cerebrospinal fluid of children with meningitis hospitalized in a tertiary hospital in Israel. Isr Med Assoc J. 2010;12(4):225–8.). However, this approach has become increasingly restricted by the elevated number of reports of resistance to β-lactam antibiotics in isolates of S. pneumoniae worldwide (²2 Waisbourd-Zinman O, Bilavsky E, Tirosh N, Samra Z, Amir J. Penicillin and ceftriaxone susceptibility of Streptococcus pneumoniae isolated from cerebrospinal fluid of children with meningitis hospitalized in a tertiary hospital in Israel. Isr Med Assoc J. 2010;12(4):225–8.). Given that the identification of a penicillin-resistant isolate of S. pneumoniae may modify the empirical antibacterial therapy, early determination of S. pneumoniae antimicrobial susceptibility is of great importance in the clinical setting (³3 Ovalle MV, Agudelo CI, Castañeda E. Empleo del disco de 1 µg de oxacilina para predecir resistencia a penicilina y ceftriaxone en Streptococcus pneumoniae. Infectio. 2001;5(3):156–61.).

The Clinical and Laboratory Standards Institute (CLSI) (⁴4 Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing. Twenty-fourth informational supplement M100-S24. Wayne, PA: CLSI; 2014.) states that the 1-µg oxacillin disk diffusion (ODD) test is an effective screening method commonly used in clinical laboratories for the detection of penicillin-resistant pneumococci. In 2008, the CLSI recommended new breakpoints for the minimum inhibitory concentrations (MICs) of penicillin and ceftriaxone, proposing different interpretive criteria for determining the use of the two antibiotics in treating meningeal and non-meningeal infections. It has been established that isolates with zones of inhibition ≥ 20 mm in the 1-µg ODD test correlate with an MIC of penicillin ≤ 0.06 µg/mL being reported as susceptible. However, in isolates with zones of inhibition ≥ 19 mm, MIC should also be determined because the zones of inhibition may be related to the level of penicillin resistance (high or intermediate) and susceptibility (⁴4 Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing. Twenty-fourth informational supplement M100-S24. Wayne, PA: CLSI; 2014.). Thus, in cases in which the zone of inhibition is ≤ 19 mm, resistance to penicillin or any other ß-lactam should not be reported before determining the MIC.

This study aimed to 1) describe the correlation between the zones of inhibition in 1-µg ODD tests and penicillin and ceftriaxone MICs of meningeal and non-meningeal strains of S. pneumoniae and 2) evaluate the usefulness of the ODD test as a predictor of susceptibility to penicillin in S. pneumoniae and as a quick and cost-effective method easily implemented in a routine clinical laboratory setting.

MATERIALS AND METHODS

S. pneumoniae isolates were obtained from a multicentric cross-sectional study conducted in various Peruvian hospitals and health centers from 2007 to 2009 (⁵5 Mercado EH, Egoavil M, Horna SG, Torres N, Velásquez R, Castillo ME, et al. Serotipos de neumococo en niños portadores antes de la vacunación antineumocócica en el Perú. Rev Peru Med Exp Salud Publica. 2012;29(1):53–60.). Healthy children 2–24 months old were enrolled in the study during their outpatient well-child visits at their respective Clinics for Growth and Development (Centros de Crecimiento y Desarrollo) or Vaccination Centers (Centros de Vacunación) (⁵5 Mercado EH, Egoavil M, Horna SG, Torres N, Velásquez R, Castillo ME, et al. Serotipos de neumococo en niños portadores antes de la vacunación antineumocócica en el Perú. Rev Peru Med Exp Salud Publica. 2012;29(1):53–60.).

All susceptibility tests were performed according to the methodology proposed by the CLSI (⁴4 Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing. Twenty-fourth informational supplement M100-S24. Wayne, PA: CLSI; 2014.). Briefly, the oxacillin (Oxoid Ltd., Hampshire, United Kingdom) disk diffusion tests were performed on a Mueller-Hinton agar supplemented with 5% defibrinated sheep blood. The isolates were incubated for 20–24 hours at 35ºC with 5%–7% CO₂. The MIC was determined using broth microdilutions of penicillin G and ceftriaxone (Sigma Aldrich Company, St. Louis, Missouri, United States), spreading 5 x 10⁵ CFU/mL onto Mueller-Hinton broth adjusted with divalent cations and supplemented with 2%–5% lysed horse blood containing double serial dilutions of the analyzed antibiotics. The MIC reading was performed after 20–24 hour incubation at 35 ºC.

The S. pneumoniae strain ATCC 49619 was used for quality control in both the ODD and MIC assays. In accordance with the CLSI recommendations, the results were interpreted using the breakpoints of penicillin and ceftriaxone for meningeal and non-meningeal strains (⁴4 Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing. Twenty-fourth informational supplement M100-S24. Wayne, PA: CLSI; 2014.). Isolates with intermediate and high levels of resistance were analyzed together as “non-susceptible” for statistical purposes.

Cohen’s kappa coefficient was used to assess the correlation between the ODD and both the penicillin MIC, using CLSI meningeal breakpoints, and the ceftriaxone MIC, using CLSI meningeal and non-meningeal breakpoints. McNemar’s chi-squared test was used to assess the correlation between the ODD and the penicillin MIC using CLSI non-meningeal breakpoints.

This study was approved by the ethics committee of the Universidad Peruana Cayetano Heredia (Lima, Peru). Informed consent for the study was obtained from the parents of the participating children. To preserve the confidentiality of patients, all participants were recorded with a study code and without personal identifiers.

RESULTS

A total of 571 S. pneumoniae strains obtained from healthy nasopharyngeal carriers less than 2 years old were analyzed. The most frequent MICs of penicillin were found to be 0.015, 0.03, and 2 µg/mL. The MIC₅₀ and MIC₉₀ of penicillin were 0.25 and 2 µg/mL respectively (Figure 1A). For ceftriaxone, the most frequent MICs were found to be 0.015, 0.03, and 1 µg/mL; the MIC₅₀ and MIC₉₀ of ceftriaxone were 0.125 and 1 µg/mL respectively (Figure 1B).

Using the MIC of penicillin and the CLSI breakpoints for meningeal strains, the rate of non-susceptible S. pneumoniae isolates was 55.0%. However, when the CLSI breakpoints for non-meningeal strains were used, the rate of non-susceptible S. pneumoniae isolates was 9.8%. The rate of non-susceptible S. pneumoniae isolates for the MIC of ceftriaxone was 21.7% and 5.8% when the CLSI breakpoints of meningeal and non-meningeal strains, respectively, were used (Figure 2).

Comparing the zones of inhibition of the ODD test and the MIC of penicillin, the use of meningeal breakpoints showed that of the 257 isolates interpreted as susceptible to penicillin based on the latter criteria (the MIC), 238 (92.6%) also demonstrated zones of inhibition ≥ 20 mm and were thus reported as susceptible to penicillin (MIC ≤ 0.06 µg/mL). A total of 19 strains (7.4%) had zones of inhibition ≤ 19 mm and were thus reported as resistant to penicillin based on the ODD test but were subsequently reported as susceptible based on the MIC of penicillin (Figure 3A). The use of meningeal breakpoints also showed that of the 314 isolates interpreted as resistant to penicillin based on the penicillin MIC criteria, 283 (90.1%) demonstrated zones of inhibition ≤ 19 mm and were thus reported as resistant to penicillin (MIC ≥ 0.12 µg/mL). A total of 31 isolates (9.9%) demonstrated zones of inhibition ≥ 20 mm and were thus reported as susceptible to penicillin based on the ODD test but resistant to penicillin based on the penicillin MIC.

Using the meningeal breakpoints, the comparison between the zones of inhibition of the ODD test and the MIC of penicillin had a good correlation value (Cohen’s kappa coefficient (k) = 0.8239). However, when the non-meningeal breakpoints were used, the correlation between the two methodologies was weak. Furthermore, ODD test zones of inhibition ≤ 19 mm with a respective resistance to penicillin (MIC ≥ 0.12 µg/mL) were found to a have a positive predictive value (PPV) of 0.937 when the meningeal breakpoints were used. In contrast, the use of non-meningeal breakpoints had a low PPV of 0.162 (Table 1).

The MIC of ceftriaxone was also compared with the zones of inhibition of the ODD test using the meningeal breakpoints (Figure 3B). Of the 447 isolates interpreted as susceptible to ceftriaxone based on its MIC, 260 (58.2%) demonstrated zones of inhibition ≥ 20 mm and were thus reported to be susceptible to ceftriaxone (MIC ≤ 0.5 µg/mL). However, 187 isolates (41.8%) demonstrated zones of inhibition ≤ 19 mm and were thus reported as resistant to ceftriaxone based on the ODD test. Using the meningeal breakpoints, 115 of the 124 isolates (92.7%) interpreted as resistant to ceftriaxone based on its MIC showed zones of inhibition ≤ 19 mm and were thus reported as resistant to ceftriaxone (MIC ≥ 2 µg/mL), whereas the remaining 9 (7.3%) showed zones of inhibition ≥ 20 mm and were thus reported as susceptible to ceftriaxone based on the ODD test.

The k values for the correlation between the ODD test and the MIC of ceftriaxone were lower than that for the MIC of penicillin when using either the meningeal or non-meningeal breakpoints (0.3352 and 0.0770 respectively). The PPV of the ODD test for resistance to ceftriaxone was found to be 0.381 when the meningeal breakpoints were used, whereas using the non-meningeal breakpoints resulted in a PPV of 0.096 (Table 2).

DISCUSSION

One of the most important findings of this study was the elevated correlation value (k = 0.8239) obtained by comparing ODD zones of inhibition ≤ 19 mm and the MIC of penicillin when the meningeal breakpoints were applied. Furthermore, comparison of these two tests showed a high PPV, providing more evidence that with proper application of meningeal breakpoints ODD tests predict S. pneumoniae resistance to penicillin almost as accurately as an MIC assessment.

Previous studies comparing ODD test zones of inhibition with MICs of penicillin and cefixime found that 98% and 99% of pneumococcal strains with no zones of inhibition were not susceptible to penicillin and cefixime respectively. Based on those results, the ODD test was used as a predictor of non-susceptibility to penicillin and cephalosporins (⁶6 Jetté LP, Sinave C. Use of an oxacillin disk screening test for detection of penicillin- and ceftriaxone-resistant pneumococci. J Clin Microbiol. 1999;37(4):1178–81., ⁷7 Doern GV, Brueggemann AB, Pierce G. Assessment of the oxacillin disk screening test for determining penicillin resistance in Streptococcus pneumoniae. Eur J Clin Microbiol Infect Dis. 1997;16(4):311–4.). It should be noted that these studies did not take into consideration whether the isolates were meningeal or non-meningeal strains when establishing the penicillin MIC breakpoints, whereas the study reported here showed that the ODD test is only a good predictor of S. pneumoniae resistance to penicillin when meningeal breakpoints are applied.

In this study, applying either the meningeal or the non-meningeal breakpoints reduced both the level of correlation between the ceftriaxone MIC and the ODD indicator of susceptibility (zones of inhibition ≤ 19 mm) and the PPV of the ODD zones of inhibition for cases of S. pneumoniae strains non-susceptible to ceftriaxone. These results contradict those from a study in Colombia that found that when the zone of inhibition of the ODD test was correlated to the pneumococcal resistance to ceftriaxone, all isolates showing some degree of resistance to ceftriaxone had a zone of inhibition of 6 mm (³3 Ovalle MV, Agudelo CI, Castañeda E. Empleo del disco de 1 µg de oxacilina para predecir resistencia a penicilina y ceftriaxone en Streptococcus pneumoniae. Infectio. 2001;5(3):156–61.). In light of the current results, ODD should not be used to predict ceftriaxone resistance. There is still no accepted disk diffusion method for detection of resistance to extended spectrum cephalosporins, mainly because of the reported severity of error (minor) of more than 15% for cefotaxime and ceftriaxone (⁸8 Weinstein MP, Klugman KP, Jones RN. Rationale for revised penicillin susceptibility breakpoints versus Streptococcus pneumoniae: coping with antimicrobial susceptibility in an era of resistance. Clin Infect Dis. 2009;48(11):1596–600.).

In the current results, which were based on isolates from Peruvian children under 2 years old, the rates of resistance to penicillin and ceftriaxone were high (55.0% and 21.7% respectively) with the use of meningeal breakpoints. In contrast, using the non-meningeal breakpoints for both antibiotics, the percentage of isolates resistant to penicillin and ceftriaxone was 9.8% and 5.8% respectively. Therefore, the strains used in this study, which were from healthy nasopharyngeal carriers, might have been causative agents of meningitis, for which high rates of resistance to penicillin have been observed. In a previous Peruvian study that tested 101 S. pneumoniae isolates causing invasive pneumococcal disease obtained from the same age group, the rates of resistance to penicillin and ceftriaxone in meningeal strains were considerably lower—34.4% and 17.4.% respectively (⁹9 Ochoa TJ, Egoavil M, Castillo ME, Reyes I, Chaparro E, Silva W, et al. Invasive pneumococcal diseases among hospitalized children in Lima, Peru. Rev Panam Salud Publica. 2010;28(2):121–7.)—but this could be attributable to the fact that nasopharyngeal strains are usually more resistant to penicillin than invasive strains. Based on these results, penicillin would not be optimal for meningeal infections but might still be the drug of choice in non-meningeal infections such as pneumonia or other non-invasive infections.

With the use of the meningeal breakpoints in isolates exhibiting ODD zones of inhibition ≤ 19 mm, this study showed that 7.4% of the isolates were susceptible to penicillin. These results concur with previous data (⁶6 Jetté LP, Sinave C. Use of an oxacillin disk screening test for detection of penicillin- and ceftriaxone-resistant pneumococci. J Clin Microbiol. 1999;37(4):1178–81.) that reported that 8.2% of S. pneumoniae strains with ODD zones of inhibition ≤ 19 mm were susceptible to penicillin (MIC ≤ 0.06 µg/mL). For isolates showing ODD zones of inhibition ≤ 19 mm, the MIC should also be determined. The reason for confirmatory testing is twofold: 1) to determine the level of penicillin resistance (intermediate or high, a distinction that is not made in the ODD test criteria) and 2) to identify penicillin-susceptible strains that showed false positives for resistance based on the ODD test (i.e., major errors) (⁷7 Doern GV, Brueggemann AB, Pierce G. Assessment of the oxacillin disk screening test for determining penicillin resistance in Streptococcus pneumoniae. Eur J Clin Microbiol Infect Dis. 1997;16(4):311–4.). Even though there is good correlation between ODD test results and penicillin MICs for penicillin resistance, using meningeal breakpoints, the use of penicillin has dropped and ceftriaxone has become the drug of choice for treatment of meningitis in many parts of the world. However, both penicillin and chloramphenicol are still considered empiric treatments for meningitis in several low-income countries (¹⁰10 Owusu M, Nguah SB, Boaitey YA, Badu-Boateng E, Abubakr AR, Lartey RA, et al. Aetiological agents of cerebrospinal meningitis: a retrospective study from a teaching hospital in Ghana. Ann Clin Microbiol Antimicrob. 2012;11:28.). Therefore, an important conclusion that can be drawn from this study, based on the high rates of penicillin resistance in meningitis that were found, is that ceftriaxone should be considered first-line empiric therapy in treatment of meningitis.

Other methodologies that can be used to complement the ODD test include the E-test, which is more affordable than MIC panels for testing penicillin and ceftriaxone. Microdilution is another reliable and relatively inexpensive method for determining MICs, but it is also cumbersome and time-consuming, and thus might not be a viable technique for analysis of outbreaks or large amounts of clinical samples. In addition, microdilution might be difficult to implement in rural areas, especially in low-income countries. Given those limitations, use of the E-test is preferable as an alternative assay to complement the ODD test (¹¹11 Rowe AK, Schwartz B, Wasas A, Klugman KP. Evaluation of the Etest as a means of determining the antibiotic susceptibilities of isolates of Streptococcus pneumoniae and Haemophilus influenzae from children in the Central African Republic. J Antimicrob Chemother. 2000;45(1):132–3.). E-test methodology can be implemented easily and has shown good correlation with the agar dilution method. When penicillin resistance rates were determined using both agar dilution and the E-test, the results had a rate of agreement of 88.6% to 92%, with no major errors (¹¹11 Rowe AK, Schwartz B, Wasas A, Klugman KP. Evaluation of the Etest as a means of determining the antibiotic susceptibilities of isolates of Streptococcus pneumoniae and Haemophilus influenzae from children in the Central African Republic. J Antimicrob Chemother. 2000;45(1):132–3., ¹²12 Mohd Nasir MD, Parasakthi N. Comparison of susceptibility test methods to detect penicillin susceptibility in Streptococcus pneumoniae isolates. Malays J Pathol. 2004;26(1):29–33.). Despite the good correlation, analysis of penicillin MICs using the E-test technique tends to result in 1 dilution lower than broth microdilution, increasing the need for extra caution when the results are within the breakpoint boundaries (¹³13 Charles MK, Berenger BM, Turnbull L, Rennie R, Fuller J. Variability of β-lactam susceptibility testing for Streptococcus pneumoniae using 4 commercial test methods and broth microdilution. Diagn Microbiol Infect Dis. 2016:84(3):240–5. doi:10.1016/j.diagmicrobio.2015.11.014.
https://doi.org/10.1016/j.diagmicrobio.2... ).

It has been proposed that susceptibility to penicillin (MIC ≤ 0.06 µg/mL) with reduced ODD zones of inhibition is related to alterations in the penicillin binding protein 2X (PBP2X) that can produce a visible effect on ODD results. The alterations would only cause a slight effect in the penicillin MICs, which would be expected to remain below the breakpoints (¹⁴14 Dowson CG, Johnson AP, Cercenado E, George RC. Genetics of oxacillin resistance in clinical isolates of Streptococcus pneumoniae that are oxacillin resistant and penicillin susceptible. Antimicrob Agents Chemother. 1994;38(1):49–53.). The exact mechanism of resistance was described in a study by Dowson et al. (¹⁴14 Dowson CG, Johnson AP, Cercenado E, George RC. Genetics of oxacillin resistance in clinical isolates of Streptococcus pneumoniae that are oxacillin resistant and penicillin susceptible. Antimicrob Agents Chemother. 1994;38(1):49–53.) in which six isolates that were susceptible to penicillin (MIC ≤ 0.06 µg/mL) had reduced ODD zones of inhibition.

Cephalosporins are known to mainly interact with the PBP2X. Consequently, the sequential acquisition of alterations in the PBPs and other genetic determinants are likely the cause of high levels of resistance to the aforementioned antimicrobial agents (¹⁵15 Fani F, Brotherton MC, Leprohon P, Ouellette M. Genomic analysis and reconstruction of cefotaxime resistance in Streptococcus pneumoniae. J Antimicrob Chemother. 2013;68(8):1718–27., ¹⁶16 Smith AM, Klugman KP. Amino acid mutations essential to production of an altered PBP 2X conferring high-level beta-lactam resistance in a clinical isolate of Streptococcus pneumoniae. Antimicrob Agents Chemother. 2005;49(11):4622–7.). These initial alterations in PBP2X are the first step to high levels of resistance to antimicrobials. Therefore, the use of cephalosporin therapy might have played a role in the creation of a selection pressure that allowed for the development of pneumococci with reduced susceptibility to oxacillin (¹⁷17 Muñoz R, Dowson CG, Daniels M, Coffey TJ, Martin C, Hakenbeck R, et al. Genetics of resistance to third-generation cephalosporins in clinical isolates of Streptococcus pneumoniae. Mol Microbiol. 1992;6(17):2461–5.).

Limitations

One limitation of this study was the use of nasopharyngeal strains of S. pneumoniae from healthy carriers. Nasopharyngeal strains are usually more resistant to penicillin than invasive strains, so the resistance percentages for the strains used in this study are most likely slightly higher than resistance percentages for invasive strains in the same population. Nevertheless, the information gathered from this study is useful for proper empiric therapy for S. pneumoniae in Latin America. Other study limitations were 1) the use of convenience sampling for selection of the health care centers and 2) the consecutive enrollment of the healthy carriers of S. pneumoniae, resulting in a sample that might not be representative of all children in Peru. However, these limitations would not have affected the correlation between the ODD zones of inhibition and the MICs.

Conclusions

Use of meningeal breakpoints resulted in good correlation between the ODD zones of inhibition and the penicillin MIC for penicillin resistance, whereas use of meningeal breakpoints for ceftriaxone, and non-meningeal breakpoints for both penicillin and ceftriaxone, did not. Therefore, the authors consider the ODD test a useful tool in predicting penicillin resistance in cases of meningeal strains of S. pneumoniae, particularly in low- and middle-income countries, where MIC determination is not routinely available.

Funding

This study was partially funded through research grants from the Instituto Nacional de Salud del Niño (MEC) and the Universidad Peruana Cayetano Heredia (TJO) (Lima, Peru).

Conflicts of interest

None.

Disclaimer

Authors hold sole responsibility for the views expressed in the manuscript, which may not necessarily reflect the opinion or policy of the RPSP/PAJPH or the Pan American Health Organization (PAHO).

REFERENCES

Publication Dates

History