Werner Ruppitsch

Classifying spa Types in Complexes Improves Interpretation of Typing Results for Methicillin-Resistant Staphylococcus aureus

ABSTRACT A total of 382 isolates of methicillin-resistant Staphylococcus aureus originating from three Austrian regions and one adjacent Italian region (Vienna, Lower Austria, North Tyrol, and South Tyrol) were typed by DNA sequence analysis of the variable repeat region of the protein A gene (spa typing). The strain collection consisted of arbitrarily chosen isolates originating from clinical specimens taken in the years 2003 to 2005 at 17 hospitals. The most common spa types found were t001 (28.8% of all isolates), t190 (27.0%), t008 (14.1%), and t041 (11.3%). The 42 remaining spa types accounted for ≤2.4% each. The dominating spa types varied between the different regions. As short sequence DNA repeat units are unstable entities, the 46 spa types were classified into seven spa complexes with respect to short sequence repeat unit composition and organization. Such classification into complexes can provide additional information for the hospital epidemiologist, empowering one to differentiate the introduction of a new strain from mere variation of endemic spa types.

Defining and Evaluating a Core Genome Multilocus Sequence Typing Scheme for Whole-Genome Sequence-Based Typing of Listeria monocytogenes

ABSTRACT Whole-genome sequencing (WGS) has emerged today as an ultimate typing tool to characterize Listeria monocytogenes outbreaks. However, data analysis and interlaboratory comparability of WGS data are still challenging for most public health laboratories. Therefore, we have developed and evaluated a new L. monocytogenes typing scheme based on genome-wide gene-by-gene comparisons (core genome multilocus the sequence typing [cgMLST]) to allow for a unique typing nomenclature. Initially, we determined the breadth of the L. monocytogenes population based on MLST data with a Bayesian approach. Based on the genome sequence data of representative isolates for the whole population, cgMLST target genes were defined and reappraised with 67 L. monocytogenes isolates from two outbreaks and serotype reference strains. The Bayesian population analysis generated five L. monocytogenes groups. Using all available NCBI RefSeq genomes (n = 36) and six additionally sequenced strains, all genetic groups were covered. Pairwise comparisons of these 42 genome sequences resulted in 1,701 cgMLST targets present in all 42 genomes with 100% overlap and ≥90% sequence similarity. Overall, ≥99.1% of the cgMLST targets were present in 67 outbreak and serotype reference strains, underlining the representativeness of the cgMLST scheme. Moreover, cgMLST enabled clustering of outbreak isolates with ≤10 alleles difference and unambiguous separation from unrelated outgroup isolates. In conclusion, the novel cgMLST scheme not only improves outbreak investigations but also enables, due to the availability of the automatically curated cgMLST nomenclature, interlaboratory exchange of data that are crucial, especially for rapid responses during transsectorial outbreaks.

Rapid identification of multidrug-resistant Mycobacterium tuberculosis isolates by rpoB gene scanning using high-resolution melting curve PCR analysis

BACKGROUND Multidrug-resistant (MDR) Mycobacterium tuberculosis poses a serious threat to the control of tuberculosis (TB) and constitutes an increasing public health problem. The availability of rapid in vitro susceptibility tests is a prerequisite for optimal patient treatment. Rifampicin resistance caused by diverse mutations in the rpoB gene is an established and widely used surrogate marker for MDR-TB. We used a high-resolution melting (HRM) curve analysis approach to scan for mutations in the rpoB gene. METHODS A total of 49 MDR-TB and 19 fully susceptible non-MDR-TB isolates, as determined by conventional drug susceptibility testing using the BACTEC-MGIT960 system, were used to evaluate the suitability of HRM curve analysis as a rapid and accurate screening system for rifampicin resistance. RESULTS HRM analysis of the rpoB cluster I site allowed the correct allocation of 44 of the 49 MDR-TB isolates and all non-MDR-TB isolates. Three of the five MDR-TB isolates (60%) falsely identified as non-MDR-TB harboured the V176F mutation that could be specifically detected by an additional HRM assay. The combined HRM analysis of all strains and isolates exhibited 95.9% sensitivity and 100% specificity. CONCLUSIONS With a positive predictive value of 100% and a negative predictive value of at least 99.9%, this combined HRM curve analysis is an ideal screening method for the TB laboratory, with minimal requirements of cost and time. The method is a closed-tube assay that can be performed in an interchangeable 96- or 384-well microplate format enabling a rapid, reliable, simple and cost-effective handling of even large sample numbers.

An outbreak of febrile gastroenteritis associated with jellied pork contaminated with Listeria monocytogenes

ZusammenfassungIm September 2008 erfuhr die Österreichische Agentur für Gesundheit und Ernährungssicherheit (AGES) von einem Ausbruch infektiöser Gastroenteritis; die Blutkultur eines 71-jährigen Hospitalisierten hatte Listeria monocytogenes erbracht. Sieben von 19 Teilnehmern eines Tagesausfluges nach Bratislava stellten Stuhlproben zur Verfügung; aus drei Proben konnte L. monocytogenes angezüchtet werden. Alle Isolate waren vom Serovar 4b und zeigten idente DNA-Fingerabdrücke. Eine Kohortenstudie zeigte, dass der Erkrankungsausbruch auf jene 16 Personen der Reisegruppe beschränkt war, die am 6. September im Rahmen eines abschließenden Heurigenbesuches zu Abend aßen. Von 15 Personen, die von einer kalten Platte aßen, erkrankten 12 (80%) binnen 24–48 h. Im Median waren die Erkrankten 62 Jahre alt. Ein 72 Jähriger erholte sich von der Gastroenteritis, wurde jedoch am Tag 19 nach dem Abendessen mit bakterieller Meningitis hospitalisiert. Die epidemiologische Abklärung belegte den Verzehr der gemischten Platte (inklusive Presswurst) als wahrscheinlichste Ursache des Krankheitsausbruchs (p = 0,0015). Diese Hypothese wurde durch den mikrobiologischen Nachweis des Erregers in der am 3. September im Heurigenbetrieb hergestellten Presswurst untermauert: L. monocytogenes fand sich in Keimzahlen von 3 × 103 bis 3 × 104 koloniebildenden Einheiten /g und war von den Humanisolaten nicht unterscheidbar. Die Symptome der 12 Patienten umfassten Fieber (12×), Durchfall (9×), Kopfweh (5×), Erbrechen (4×), Gliederschmerzen (2×), und Halsweh (1×). Die aktive Fallsuche erbrachte zudem einen Fall von Rhombencephalitis (weiblich, 48 Jahre alt); von einer 4-Personengruppe hatten am 6. September nur die Patientin und ihr asymptomatischer Gatte saure Presswurst mit Zwiebel konsumiert. Bislang war in Österreich ein Ausbruch von L. monocytogenes-assozierter Gastroenteritis noch nicht beschrieben worden. Das Auftreten von eitriger Meningitis (diagnostiziert am Tag 19 nach Verzehr von kontaminierter Presswurst) bei einem Durchfallpatienten belegt ein signifikantes Risiko für systemische Listeriose bei älteren Patienten mit febriler Gastroenteritis durch L. monocytogenes; eine antibiotische Therapie sollte in derartigen Fällen gesicherter Listerien-Gastroenteritis in Betracht gezogen werden.SummaryIn September 2008, the Austrian Agency for Health and Food Safety (AGES) learned of an outbreak of diarrheal illness that included a 71-year-old patient hospitalized for gastroenteritis with a blood culture positive for Listeria monocytogenes. Three stool specimens provided by seven of 19 persons attending a day trip to a foreign city, including a final break at an Austrian tavern, yielded L. monocytogenes. All isolates were of serovar 4b and had fingerprints indistinguishable from each other. A cohort study revealed that the outbreak of gastroenteritis occurred among 16 persons who had eaten dinner at the wine tavern on September 6. Of the 15 persons who ate from platters of mixed cold-cuts, 12 (80%) developed symptoms of febrile gastroenteritis within 24–48 h. The median age of those who became ill was 62 years. A 72-year-old patient recovered from gastroenteritis but was hospitalized with bacterial meningitis on day 19 after the dinner. The epidemiological investigation identified the consumption of mixed cold-cuts (including jellied pork) at the wine tavern as the most likely vehicle of the foodborne outbreak (P = 0.0015). This hypothesis was confirmed by microbiological investigation of jellied pork produced by the tavern owner on September 3. L. monocytogenes was isolated from leftover food in numbers of 3 × 103–3 × 104 colony forming units/g and was indistinguishable from the clinical outbreak isolates. Symptoms reported by the 12 patients included unspecified fever (12×), diarrhea (9×), headache (5×), vomiting (4×), body aches (2×) and sore throat (1×). Active case finding identified one case of rhombencephalitis (female, age 48) among another group of four guests, among whom only the patient and her asymptomatic husband had eaten jellied pork on September 6. This is the first outbreak of L. monocytogenes-associated gastroenteritis reported in Austria. The occurrence of a secondary case of meningitis (diagnosed on day 19 after consumption of jellied pork) indicates a significant risk of systemic listeriosis among elderly patients with febrile gastroenteritis caused by L. monocytogenes; antibiotic therapy should therefore be considered in such cases of documented listerial gastroenteritis.

Outbreak of staphylococcal food intoxication after consumption of pasteurized milk products, June 2007, Austria

ZusammenfassungAm 13. Juni 2007 wurde Österreichische Agentur für Gesundheit und Ernährungssicherheit von einer lokalen Gesundheitsbehörde über das gehäufte Auftreten von Bauchkrämpfen und Erbrechen am 8. Juni bei 40 Kindern zweier benachbarter Volksschulen unterrichtet. Die am 8. Juni konsumierten Schulmilchgetränke wurden als Quelle des Ausbruchs vermutet. Die Schulmilchgetränke stammten von einer lokalen Molkerei X, die am 8. Juni acht Volksschulen und zwei Kindergärten belieferte. Die kurze Inkubationszeit – alle Fälle erkrankten noch am Tag der Konsumation – und die kurze Krankheitsdauer von 1–2 Tagen ließen eine Lebensmittelvergiftung vermuten. Deskriptiv-epidemiologische und mikrobiologische Untersuchungen sowie eine retrospektive Kohortenstudie wurden zur Ermittlung des ursächlichen Agens, dessen Reservoirs und des Übertragungsmodus durchgeführt.Von 1025 Kindern der 10 Einrichtungen erfüllten 166 die Falldefinition (16,2%). Nach den Ergebnissen der Kohortenanalysen war die Konsumation von Milch, Kakaomilch oder Vanillemilch der Molkerei X mit einem 37,8 fach höherem Risiko zu erkranken, assoziiert (95% CI: 2,3–116,5). In den verbliebenen orginal-verschlossenen Milchprodukten wurden Staphylokokken-Enterotoxin A und D nachgewiesen. Sechs von 64 Viertelgemelksproben von 3 von 16 der Molkerei X zuliefernden Kühen erbrachten in der bakteriologischen Untersuchung Enterotoxin und D produzierenden Staphylococcus aureus des spa Typs t2953. Ein aus einem Nasenabstrich des Molkereibetreibers gewonnenes S. aureus Isolat war vom spa Typ t635 und wies Gene für Enterotoxin C, G, H und I auf. Die Ausbruchsuntersuchung belegte, dass die am 7. Juni in der Molkerei X hergestellten Milchprodukte die Quelle des Ausbruches einer Staphylokokken-Enterotoxikose am 8. Juni waren. Die Kühe der Molkerei X – und nicht der Molkereibetreiber – waren das wahrscheinliche Reservoir des Enterotoxin-produzierenden S. aureus-Stammes. Eine Risikoanalyse des Produktions-Prozesses ließ vermuten, dass die Toxinproduktion während Lagerung von überschüssiger pasteurisierter Milch über 3 Tage stattfand, bevor diese am 7. Juni neuerlich pasteurisiert und zu den inkriminierten Schulmilchgetränken verarbeitet wurde.SummaryOn June 13, 2007, the public health authority informed the Austrian Agency for Health and Food Safety about 40 children from two neighboring elementary schools who had fallen ill with abdominal cramps and vomiting on June 8. School milk products consumed on June 8 were suspected as the source of the outbreak. On June 8, the milk products provided by local dairy X to eight elementary schools and two nurseries. The short incubation period – all cases fell ill on the day on which the products were consumed – and the short duration of illness (1–2 days) strongly suggested intoxication. In order to identify the causative pathogen, its reservoir and the mode of transmission, a descriptive-epidemiological and microbiological investigation and a retrospective cohort study were conducted. Six of the 10 institutions served by dairy X completed questionnaires on demographics and food consumption. One school had a 79% response rate (203/258) and was chosen as the basis for our cohort study. A total of 166 of the 1025 children (16.2%) at the 10 institutions fulfilled the case definition. Consumption of milk, cacao milk or vanilla milk originating from dairy X was associated with a 37.8 times higher risk of becoming a case (95% CI: 2.3–116.5). Unopened milk products left over at the affected institutions yielded staphylococcal enterotoxins A and D. Six out of 64 quarter milk samples from three of 16 cows producing milk for dairy X tested positive for S. aureus. The isolates produced enterotoxins A and D, yielded genes encoding enterotoxins and D, and showed spa type t2953. S. aureus isolated from the nasal swab of the dairy owner harbored genes encoding enterotoxins C, G, H and I, and showed spa type t635. Our investigation revealed that the milk products produced in dairy X on June 7 were the source of the outbreak on June 8. The cows – not the dairy owner – the likely reservoir of the enterotoxin-producing S. aureus. From the risk assessment of the production process at the dairy, we hypothesize that staphylococcal toxin production took place during a 3-day period of storage of pasteurized milk prior to repasteurization for the production batch of 7.

Methicillin-resistant Staphylococcus aureus: a new zoonotic agent?

ZusammenfassungStaphylococcus aureus ist ein bedeutender Erreger von Infektionen, die in Krankenhäusern und Pflegeeinrichtungen erworben werden, und bei der Allgemeinbevölkerung. Ein Drittel der Gesamtbevölkerung ist mit Staphylococcus aureus kolonisiert, wobei das Risiko, an einer Infektion durch Staphylococcus aureus zu erkranken bei diesen kolonisierten Personen erhöht ist. Die weltweit zunehmende Antibiotikaresistenz von Staphylococcus aureus schränkt die Behandlungsmöglichkeiten von Infektionen ein und erschwert Kontrollmaßnahmen. Kurz nach Einführung des Methicillins wurde über die ersten Methicillin-resistenten Staphylococcus aureus Isolate (MRSA) berichtet. Während des letzten Jahrzehnts vollzog sich außerdem eine epidemiologische Verschiebung von den bisher dominierenden Krankenhaus assoziierten MRSA Stämmen hin zu in der Gemeinschaft erworbenen MRSA Stämmen (community acquired MRSA, CA-MRSA), welche Infektionen auch bei dem Teil der Bevölkerung hervorrufen, der keine Risikofaktoren durch Krankenhauskontakte hat. Basierend auf neueren Untersuchungen besteht für den Sequenztyp (ST) 398 eine kausale Verbindung zwischen einer MRSA Kolonisation im Viehbestand und der MRSA Besiedlung, sowie dem Auftreten von Infektionen, bei Tierzüchtern. In den Niederlanden wurde eine hohe Besiedlungsrate durch den MRSA ST398 bei Schweinen und Schweinehaltern festgestellt. Weltweite Berichte über den Nachweis von ST398 MRSA verdeutlichen, dass das Auftreten des neuen Subtyps kein spezifisch holländisches Problem darstellt. In Österreich konnte der MRSA Sequenztyp 398 in Staubproben von Schweinezuchtbetrieben und aus Lebensmitteln isoliert werden. Seit dem ersten Auftreten des Sequenztyps ST398 als Infektionserreger beim Menschen in Österreich im Jahre 2006 konnten 21 Humanisolate nachgewiesen werden, wovon allerdings nur ein geringer Teil mit Infektionen assoziiert war. MRSA müssen zukünftig auch als zoonotisches Agens betrachtet werden, wobei der Nutztierbestand ein bedeutendes Reservoir darstellt. Weitergehende Untersuchungen sind nötig, um Kontrollmaßnahmen erarbeiten und implementieren zu können.SummaryStaphylococcus aureus is a major cause of infection in hospitals and the community. One third of the general population is colonized by the bacterium, constituting a risk factor for acquisition of infection with this pathogen. Worldwide, the increasing antibiotic resistance of S. aureus complicates treatment of infection and control measures. Soon after the introduction of methicillin, the first isolates resistant to this antibiotic were reported and named methicillin-resistant S. aureus (MRSA). During the past decade a major change in MRSA epidemiology has been observed: whereas in the past MRSA was almost exclusively regarded a hospital pathogen, the advent of community-acquired MRSA has led to infections in people without hospital-related risk factors. Recent evidence has also identified a link between colonization of livestock and MRSA carriage and infections in people who work with animals. Screening of pigs and pig farmers in the Netherlands revealed high prevalence of MRSA sequence type (ST) 398 and it has become clear that the emergence of ST398 is not just a Dutch problem, as reports on livestock colonization and human infections are appearing worldwide. In Austria, the ST398 lineage has been detected in dust samples from pig breeding facilities and in food samples. Since the first Austrian detection of this emerging lineage in 2006, 21 human isolates, partially associated with infections, have been observed. MRSA has to be regarded as a new emerging zoonotic agent and livestock may constitute a growing reservoir of the ST398 lineage. More information is needed so that control measures to reduce the impact of the emerging MRSA ST398 lineage on public health can be developed and implemented.

Occurrence of Vibrio cholerae serogroups other than O1 and O139 in Austria

ZusammenfassungVibrio cholerae non-O1/non-O139 können weltweit verbreitet sowohl im Süßwasser als auch im Brackwasser angetroffen werden. Besonders in der warmen Jahreszeit können sie sich zu Konzentrationen vermehren, die für das Zustandekommen von Infektionen relevant sind. In den Jahren 2000 bis 2005 wurden in Österreich 13 Infektionen mit non-O1/non-O139 Vibrio cholerae dokumentiert. Zwölf Patienten (im Alter von 8 bis 65 Jahren; 7 männlich) erkrankten manifest: Durchfall × 5, Otitis × 6, Sepsis × 1. Alle 5 Patienten, deren Infektionen auf Auslandsaufenthalte zurückgeführt werden konnten, hatten Diarrhoe. Die 8 Personen ohne Auslandsanamnese hatten Otitis media (n = 4), Otitis externa (n = 2), Sepsis (n = 1) und einmal einen asymptomatischen Verlauf. Die tödlich verlaufende Sepsis betraf einen Fischer vom Neusiedler See eine Woche nach Abschluss einer Chemotherapie (Plasmozytom). Detaillierte Reiseanamnesen innerhalb Österreichs waren für 5 der 8 Personen erhältlich: für alle Fünf waren Aufenthalte am Neusiedler See belegbar. Warum sich die im Inland erworbenen Infektionen vorwiegend als Otitis und nicht als Diarrhoe manifestieren ist unklar. Wir nehmen an, dass Durchfallerkrankungen durch non-O1/non-O139 Vibrio cholerae in Österreich aufgrund der Vorgehensweisen in mikrobiologischen Laboren nicht erkannt werden. Eine bakteriologische Untersuchung auf Vibrio cholerae erfolgt in Regelfall nur bei entsprechender Auslandsanamnese.SummaryFrom 2000 to 2005, 13 infections due to non-O1/non-O139 Vibrio cholerae were documented in Austria. Twelve patients (8 years to 65 years old; 7 male) had symptomatic infections: diarrhea × 5, otitis × 6, septicemia once. All 5 patients who acquired their infections abroad, suffered from diarrhea. The 8 persons without travel history outside of Austria had otitis media (n = 4) or otitis externa (n = 2); the lethal case of septicemia affected a fisherman with underlying malignancy. One isolate was from an asymptomatic child. Detailed data on travel history inside Austria was available for 5 of these 8 patients: all 5 had visited or lived near Austrias largest lake. The concentration of salt in this westernmost steppe lake in Europe is approximately one-twentieth of that of sea water. Why otitis and not diarrhea is the dominating manifestation of non-O1/non-O139 infection acquired in Austria remains to be elucidated. We hypothesize that diarrhea due to Vibrio cholerae serogroups other than O1 and O139 acquired in Austria may simply be unrecognized by the standard operating procedures employed in clinical microbiology laboratories. Testing for Vibrio cholerae is not considered necessary for domestically acquired diarrhea. Only in patients who acquired diarrhea abroad, do physicians sometimes consider cholera as a differential diagnosis, thereby prompting the laboratory to use thiosulfate citrate bile salt sucrose (TCBS) agar plates.

Gene Scanning of an Internalin B Gene Fragment Using High-Resolution Melting Curve Analysis as a Tool for Rapid Typing of Listeria monocytogenes

The ability to accurately track Listeria monocytogenes strains involved in outbreaks is essential for control and prevention of listeriosis. Because current typing techniques are time-consuming, cost-intensive, technically demanding, and difficult to standardize, we developed a rapid and cost-effective method for typing of L. monocytogenes. In all, 172 clinical L. monocytogenes isolates and 20 isolates from culture collections were typed by high-resolution melting (HRM) curve analysis of a specific locus of the internalin B gene (inlB). All obtained HRM curve profiles were verified by sequence analysis. The 192 tested L. monocytogenes isolates yielded 15 specific HRM curve profiles. Sequence analysis revealed that these 15 HRM curve profiles correspond to 18 distinct inlB sequence types. The HRM curve profiles obtained correlated with the five phylogenetic groups I.1, I.2, II.1, II.2, and III. Thus, HRM curve analysis constitutes an inexpensive assay and represents an improvement in typing relative to classical serotyping or multiplex PCR typing protocols. This method provides a rapid and powerful screening tool for simultaneous preliminary typing of up to 384 samples in approximately 2 hours.

One-Step Triplex High-Resolution Melting Analysis for Rapid Identification and Simultaneous Subtyping of Frequently Isolated Salmonella Serovars

ABSTRACT Salmonellosis is one of the most important food-borne diseases worldwide. For outbreak investigation and infection control, accurate and fast subtyping methods are essential. A triplex gene-scanning assay was developed and evaluated for serotype-specific subtyping of Salmonella enterica isolates based on specific single-nucleotide polymorphisms in fragments of fljB, gyrB, and ycfQ. Simultaneous gene scanning of fljB, gyrB, and ycfQ by high-resolution melting-curve analysis of 417 Salmonella isolates comprising 46 different serotypes allowed the unequivocal, simple, and fast identification of 37 serotypes. Identical melting-curve profiles were obtained in some cases from Salmonella enterica serotype Enteritidis and Salmonella enterica serotype Dublin, in all cases from Salmonella enterica serotype Ohio and Salmonella enterica serotype Rissen, from Salmonella enterica serotype Mbandaka and Salmonella enterica serotype Kentucky, and from Salmonella enterica serotype Bredeney, Salmonella enterica serotype Give, and Salmonella enterica serotype Schwarzengrund. To differentiate the most frequent Salmonella serotype, Enteritidis, from some S. Dublin isolates, an additional single PCR assay was developed for specific identification of S. Enteritidis. The closed-tube triplex high-resolution melting-curve assay developed, in combination with an S. Enteritidis-specific PCR, represents an improved protocol for accurate, cost-effective, simple, and fast subtyping of 39 Salmonella serotypes. These 39 serotypes represent more than 94% of all human and more than 85% of all nonhuman Salmonella isolates (including isolates from veterinary, food, and environmental samples) obtained in the years 2008 and 2009 in Austria.

Limited value of routine spa typing: A cross-sectional study of methicillin-resistant Staphylococcus aureus-positive patients in an Austrian hospital

BACKGROUND We investigated whether spa typing is useful for indicating the setting of methicillin-resistant Staphylococcus aureus (MRSA) acquisition (community or health care acquired), the clinical relevance (colonization or infection), the type of infection (invasive or noninvasive), and the clinical outcome. METHODS Between August 2006 and December 2009, 381 routinely diagnosed culture-confirmed MRSA-positive patients were included into a cross-sectional study at an 800-bed hospital. RESULTS Out of 159 patients with colonization, 27 (17%) acquired MRSA in the community (CA-MRSA) and 123 (77.4%) in health care settings (HA-MRSA), and, of the 222 patients with infections, 119 (53.6%) had HA-MRSA and 103 (46.4%) had CA-MRSA. The 10 most frequent spa types accounted for 68.2% of the 346 typed MRSA isolates: t190 (28.3%), t032 (16.5%), t041 (9.4%), t008 (8.4%), t001 (3.4%), t002 (2.9%), t044 (3.1%), t223 (2.1%), t015 (2.1%), t127 (1.3%). CONCLUSION Spa typing of routinely identified MRSA isolates is unsuitable to predict the likeliness of an infection, of an invasive infection, and the clinical outcome. Molecular criteria such as spa type or Panton-Valentine leukocidin positivity used for classifying MRSA as either belonging to a community or hospital clone are of limited value to indicate the setting, where the MRSA strain was actually acquired according to epidemiologic criteria.