Blood, liver, and skin samples sent to the Centers for Disease Control and Prevention (CDC; Atlanta, GA, USA) arrived on October 10. The following day the Infectious Disease Pathology Branch reported that immunohistochemical staining of liver and skin sections resulted in the detection of antigen with monoclonal antibody that was broadly cross-reactive for Old World arenaviruses, the first indication of an etiologic diagnosis (, panel B). Consequently, the same arenavirus RT-PCR procedures as above were applied to liver extracts and produced positive results at NICD and the Special Pathogens Branch, CDC. One RT-PCR procedure yielded ≈300-bp glycoprotein gene product (1
), and the other yielded ≈1,000-bp nucleoprotein gene product (2
). Nucleotide sequencing of the PCR products and phylogenetic analysis performed as described (3
) but using MEGA 4.0 (4
) showed that a novel arenavirus, since named Lujo virus (5
), was involved ().
Figure 3 Neighbor-joining tree reconstructed by using bootstrap analysis with 1,000 pseudoreplicate datasets showing the phylogenetic relationship of known arenaviruses (data derived from GenBank) to the novel Lujo arenavirus from southern Africa (boldface), inferred (more ...)
Replicate nucleic acid extracts from liver samples that were sent to Columbia University (New York, NY, USA) were subjected to 454 pyrosequencing to produce full-length genome sequences of the arenavirus, extending the preliminary findings based on partial sequence data to indicate that the new virus is related to, but distinct from, known Old World arenaviruses (5
). The novel arenavirus was isolated in culture from the blood and liver samples of patients 2 and 3, as confirmed by immunofluorescence with polyvalent Old World arenavirus antiserum and sequencing of PCR products. However, the isolates from the blood samples took a minimum of 7 days to become detectable in culture, as compared with 3–5 days for liver samples and other blood samples tested during the investigation. Thus, it can be surmised that the viral loads were likely too low in the blood samples of patients 2 and 3 for detection of viral nucleic acid with the generic primers.
Through contact tracing, we found that a cleaner who worked at the hospital where patient 1 was treated, patient 4 (), became ill on September 27, was admitted to a provincial hospital on October 5, and was transferred the same day to a tertiary academic hospital. She had a chronic underlying disease and died on the day after admission. Her only potential exposure to infection occurred September 14 when she cleaned the cubicle where patient 1 was treated. On October 9, a second nurse, patient 5 () became sick and was admitted to the hospital the following day. She had attended patient 2 and been involved in the insertion of a central venous line on September 27 before barrier nursing was instituted.
Treatment of patient 5 with the oral form of the antiviral drug ribavirin was started on October 11 with a loading dose of 30 mg/kg (2 g), followed by a projected schedule of 15 mg/kg every 6 h (1 g) for 4 days and 7.5 mg/kg every 8 h for 6 days. However, after the patient was intubated on October 12, treatment was continued through nasogastric tube until the intravenous formulation of the drug was obtained on October 17 (6
). The patient reported improvement beginning October 27 and was discharged from the hospital on December 2 after RT-PCR did not detect viral RNA in blood and urine on 3 consecutive occasions.
The first 4 patients were initially managed without special infection control precautions, apart from the donning of surgical gloves for intubation or the taking of blood samples, and the use of impervious aprons in wards. Consequently, healthcare workers and maintenance staff such as cleaners were all potentially exposed to contaminated bedding, eating utensils, and bedpans which contained excreta and vomitus. On the other hand, no incidents occurred that could be construed as constituting specific exposure to infection, for example, needlesticks. After the possible involvement of a viral hemorrhagic fever was recognized on September 28 following the admission of patient 2 to the hospital in Johannesburg, barrier nursing procedures were instituted as successive patients were identified, but only patient 5 was managed with full precautions throughout her illness. The full precautions consisted of the donning of double surgical gloves, protective oversuits, impervious overshoes, disposable balaclavas, and N95 masks plus goggles or visors. Alternatively, the protective oversuits were replaced with surgical scrub-suits and impervious disposable gowns. No infections appeared to have occurred after the adoption of the full precautions.
The putative incubation periods for patients 2 to 5 ranged from 7 to 13 days, and the periods of illness for the 4 patients with fatal infections ranged from 10 to 13 days (). All patients sought treatment for nonspecific febrile illness with headache and myalgia, which increased in severity over 7 days with the development of diarrhea and pharyngitis. A morbilliform rash became evident on the face and trunk in 3 Caucasian patients on day 6–8 of illness, but not in 2 African patients, and neck and facial swelling occurred in 3 patients. Four patients exhibited transient subjective improvement after ≈1 week of illness; this brief improvement was followed by rapid deterioration with respiratory distress, neurologic signs, and circulatory collapse, the terminal features in patients who died. Bleeding was not a prominent feature. However, 1 patient had a petechial rash, 1 had gingival bleeding, and another experienced the oozing of blood from venipuncture sites. Chest pain was a prominent symptom for 2 patients. All patients had thrombocytopenia on admission to the hospital (platelet count range 20–104 × 109 cells/L). Three patients had leukocyte counts in the normal range, and 2 had leukopenia on admission, whereas leukocytosis developed in 4 patients during the illness. The maximum aspartate aminotransferase values recorded in the 4 patients who died from the disease ranged from 549 IU/L to 2,486 IU/L, compared with 240 IU/L in the survivor who was treated with ribavirin.
Blood samples from patients 4 and 5 were positive by RT-PCR for arenavirus nucleic acid at NICD and yielded virus in culture. Blood samples taken from patient 1 on September 12 and from patient 2 on September 25 were traced to a hospital laboratory in Lusaka and sent to CDC late in the course of the investigation. The sample from patient 1 was RT-PCR positive, and both samples yielded isolates of the novel virus in cell culture. In summary, isolation of virus was achieved from the blood samples of all 5 patients, plus liver samples from patients 2 and 3, at NICD, CDC, or both ().
Summary of diagnostic RT-PCR and virus isolation studies on 5 novel arena virus–infected patients, southern Africa, 2008*
Tracing and monitoring of all contacts of known patients for 21 days from last date of contact with a patient or fomites in Zambia and South Africa failed to identify cases additional to those reported here. Antibody surveys still need to be conducted to assess the occurrence of less severe infections and to determine the distribution and prevalence of the virus. Rodent studies are also indicated, particularly in the areas frequented by patient 1, as the original source of the infection remains unknown.