Investigative Plans

At the request of Congolese officials, World Health Organization (WHO) response teams intervened in both outbreaks. However, because of a delayed alert and bureaucratic challenges, the teams arrived 2 months after onset of the epidemics, which limited the number of case-patients who were available for clinical examination and appropriate sampling. As a result of the circumstances, the following investigative plan was established.

The suspected plague deaths that occurred before the WHO response team arrival were attributed to plague based on the epidemiologic context, the reported clinical signs, and the rapid disease onset typically observed for pneumonic plague. However, no postmortem specimens could be taken for confirmation. Suspected convalescents were tracked, interviewed, and blood samples were collected in standard serologic tubes by a clinician and stored at 4°C for retrospective serologic testing.

Each new patient with clinically suspected pneumonic plague was admitted to an isolation center to receive the first doses of the national protocol treatment. Blood was immediately sampled and sputum samples were collected by a trained biologist from the National Plague Reference Laboratory who supported the WHO mission. Sampling kits, designed by the Institut Pasteur de Madagascar (IPM; Antananarivo, Madagascar) for improving laboratory confirmation of plague cases in this country, were used to collect and prepare sputum samples for transport. The kits contained the necessary equipment for sampling either sputum or bubo aspirate and provided an effective method for collecting pure samples with minimal chance of contamination. One kit was required per patient; each contained a sterile syringe for sample extraction from a bubo or sputum sampling cup, an Eppendorf tube containing 1 mL of phosphate-buffered saline (PBS) for sample dilution, a culture tube with Cary Blair (CB) medium, a sterile swab for placement of pure sample into CB media for transport, a sterile test tube for RDT, isopropyl alcohol and antiseptic towelettes, and a patient record form. Safety procedures included use of proper personal protective equipment and appropriate waste management.

Specimen Handling and Transportation

Figure 2

Figure 2. Flow of sample processing for specimens for pneumonic plague outbreaks in Zobia, Democratic Republic of the Congo (DRC), 2005 (A), and Bolebole, DRC, 2006 (B). PBS, phosphate-buffered saline; RDT, rapid diagnostic...

In the 2005 Zobia outbreak, handling and transportation of the specimens were planned as follows. Sputum specimens were immediately evaluated on site by a biologist of the National Plague Reference Laboratory using direct microscopy and RDT. The specimens were then transported to a temporary second-line regional laboratory in Kisangani, DRC, by air (2 hours) on roughly a weekly basis. In Kisangani, a biologist from IPM who was assigned to the WHO mission performed culture identification for Y. pestis and direct microscopy and prepared specimens for the twice weekly air shipment (>1,000 km) to the Institut National pour la Recherche Biologique in Kinshasa, DRC. At the time of arrival at Institut National pour la Recherche Biologique, specimens were further distributed by commercial air carriers to IPM in Antananarivo, Madagascar; the National Institute for Communicable Diseases (NICD) in Johannesburg, South Africa; and the Centre de Recherche du Service de Santé des Armées in Grenoble, France, for additional analyses and serologic testing (Figure 2, panel A).

In the 2006 Bolebole outbreak, a streamlined strategy was defined to limit sample passages and delays in transport. Emphasis was placed on RDT and direct microscopy on site, but no other manipulation of samples was allowed. Regional and national steps were bypassed and samples were sent directly to the international reference laboratory at IPM, where confirmatory tests were performed (Figure 2, panel B).

Specimen Analyses

Sputum smears were stained (Gram and Wayson) and directly examined by microscopy according to standard protocols (16). In addition, sputum was extracted from swabs stored in CB by using 1 mL of PBS and cultured by using standard methods for plague isolation as recommended by WHO (16). Suspect colonies were confirmed by using standard biochemical tests and phage lysis as described (16–19). The above methods were performed at Kisangani, NICD, and IPM during the first outbreak and only at IPM during the second outbreak.

RDT for Y. pestis specific F1-antigen detection by immunochromatography was performed at the frontline. The dipstick is contained in an individually vacuum-sealed package with desiccant to maintain stability and sterility and is used with the sampling kit described above. The specimen was processed by using 0.5 mL of thick sputum diluted in 1 mL of PBS and homogenized by using a sterile syringe. A 200-μL aliquot of homogenate was placed in a sterile test tube and the dipstick applied. The test was considered positive when 2 pink lines (control line + test line) appeared after 15 minutes as described (18). To simplify the procedures in the field, semiquantitative grading was not requested.

One serum sample was drawn as early as possible after onset of symptoms and a second serum sample 10 days later to allow for the development of an antibody response. A positive result was determined by a 4-fold increase in the antibody titer from the first to the second sample (1). Serum was transported at 4°C or at ambient temperature and filter-sterilized upon arrival. ELISA detecting anti-F1 immunoglobulin (Ig) G was performed as previously described (20) at Centre de Recherche du Service de Santé des Armées.

Direct fluorescent antibody (DFA) staining was performed at NICD on sputum specimens from the first outbreak according to standard protocols (16,21,22). Smears were a thin impression (touch preparation) of sputum on swabs transported in CB medium. The presence of plague bacilli was tested through the direct binding of fluorescein isothiocyanate–conjugated F1 antibody to antigen. A smear was positive when bright, intense green staining around bacteria was observed.

Specimen Handling and Transport

Pneumonic plague was suspected late after the onset of the outbreak in Zobia. No clinical specimen had been taken before the arrival of the WHO response team because first-line staff had not been properly trained and appropriate sampling materials were not available. After deployment, and under direct management of the response team, the frontline laboratory conducted RDT and direct microscopy; results indicated plague. All patients with suspected plague were subsequently isolated and treated, and close contacts received chemoprophylaxis as recommended by WHO (1). During this outbreak, difficulties with isolating Y. pestis were anticipated and a second-line laboratory in Kisangani was established. Unfortunately, the functions of this laboratory were limited by power and water shortages, which restricted the capacity to perform. As such, this laboratory in practice served only as a logistic platform for the air shipment of specimens to Kinshasa. Shipments from Zobia to Madagascar took 8–40 days (median 18.5 days). Sputum samples in CB were stored at 28°C–30°C, and serum was stored at 4°C.

At Bolebole, greater emphasis was placed on front-line sampling, avoiding dilutions, and multiple handling of specimens. Only direct microbiology and RDT for F1 detection were conducted on site, which provided results indicating plague. Samples were then obtained by specimen collection kits and sent in good condition. Samples were stored at the same temperatures as during the first outbreak, were transported once a week by road from Bolebole to Isiro (2 days), and then shipped by air to Kinshasa on an 8-hour flight with 3 intermediate landings. Finally, specimens were shipped to Madagascar weekly by commercial airlines. In total, the transport of samples from Bolebole to IPM took 4–5 days.

Use of RDT

Cost-effective, easy to use, and reliable, the RDT can be considered as an alert tool. It has proven to be rapid and easy to use at the bedside with 100% specificity and sensitivity (18). Importantly, detection of the F1-antigen is possible even in cases that have been previously treated, which was common for many of the patients in the outbreaks we describe. However, the RDT is not a screening test and must be used only to confirm a clinical suspicion. The RDT is also helpful for outbreak control. In both Zobia and Bolebole, one of the most challenging and time-consuming activities was managing the close contacts (≈25 for each suspected case). Even though the RDT results have no confirmatory value during the early phase of the outbreak, they enable management of close contacts to be focused around cases defined as probable within 15 minutes. Notably, the RDT becomes a field confirmation test after Y. pestis has been identified on site (7).

Specimen Collection and Testing

In addition to sputum samples, a serum sample was drawn from all patients admitted to the isolation center as well as from convalescents. However, in circumstances as described above, it was challenging to obtain a second blood sample from the patients 10 days later. As for the suspect cases that occurred before the arrival of the response team, motivation for testing was weak and no suspect patients returned. This was because most patients were young male adults, who returned to mining activities after cure and release from the isolation center. Any kind of control was perceived only as a limitation to business as usual. Overall, only 6 pairs of serum samples were obtained from patients treated at an early stage in Zobia, despite a strong emphasis being placed on serology for confirmation. The situation was worse in Bolebole, as no paired serum samples were obtained.

Isolation of Y. pestis from sputum was desired but challenging because of administration of antimicrobial drugs to patients before specimen collection, the nonsterile nature of the specimen, the slow growth of Y. pestis in culture, and prolonged specimen handling and transport. In addition, results from DFA and real-time PCR testing may also have been affected by prolonged handling and transport.

Because of the limitations in the field, it is difficult to extract information in regard to the respective performances of different diagnostic techniques. RDT is an established test for Africa and is known for its high sensitivity and specificity. If nearly half of the tested patients were positive in Zobia and 24% in Bolebole (Table), this can be due to inappropriate specimens (saliva instead of sputum) or a misdiagnosis. However it should be noted that 4 of 5 patients with confirmed plague in Zobia had a positive RDT result (DFA analyses were positive for 2 of them). In Bolebole, the 4 patients with plague confirmed by culture also had a positive RDT result.

Scope of the Outbreak

The primary objective of the laboratory investigation was to confirm the existence of ongoing pneumonic plague in the communities, to set up an ad hoc control strategy, and to stop the spread of disease. With regard to the magnitude of the 2 outbreaks, one cannot discard the possibility that a certain number of reported cases were not due to plague. However, one cannot discard the possibility that several plague cases were neither diagnosed nor reported. This is particularly true for the period that preceded the international intervention. In Zobia, to be as specific as possible, only the patients who died of a short-course and severe febrile pneumonic syndrome at this period were considered to have died of plague. But what was the number of survivors? This is difficult to project. The case-fatality ratio observed during the mission (11.0%) cannot be applied at the preintervention period because case management had greatly improved. In addition, the peak of the outbreak had passed, and the number of true plague cases among those clinically detected was probably lower because less than half of the patients with acute illness had a positive RDT (Table). The situation was similar at Bolebole.