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Dispatch
Polymerase Chain Reaction
Assay and Bacterial Meningitis Surveillance in Remote Areas, Niger
Fati Sidikou,* Saacou Djibo,* Muhamed Kheir Taha,† Jean Michel Alonso,†
Ali Djibo,‡ Kiari Kaka Kairo,‡ Suzanne Chanteau,* and Pascal Boisier*
*Centre de Recherches Médicales et Sanitaires, Niamey, Niger; †Institut
Pasteur, Paris, France; and ‡Ministère de la Santé, Niamey, Niger
Suggested citation
for this article:
Sidikou F, Djibo S, Taha MK, Alonso JM, Djibo A, Kairo KK, et al. Polymerase
chain reaction assay and bacterial meningitis surveillance in remote
areas, Niger. Emerg Infect Dis [serial online] 2003 Nov [date cited].
Available from: URL: http://www.cdc.gov/ncidod/EID/vol9no11/03-0462.htm
To compensate for
the lack of laboratories in remote areas, the national reference laboratory
for meningitis in Niger used polymerase chain reaction (PCR) to enhance
the surveillance of meningitis caused by Neisseria meningitidis,
Streptococcus pneumoniae, and Haemophilus influenzae.
PCR effectively documented the wide geographic spread of N. meningitidis
serogroup W135.
Niger is a sub-Saharan country located in the center of the African “meningitis
belt,” which includes all or parts of 18 sub-Saharan countries (1).
The population of approximately 11 million is mainly concentrated in the
southern part of the territory; 84% of the population lives in rural areas.
Every year, during the dry, warm, and windy season, between January and
May, localized epidemics of meningococcal meningitis occur in several
districts, whereas major epidemics, affecting the entire territory, break
out at regular intervals of every few years (2,3). Previously,
all important epidemics have been caused by Neisseria meningitidis
serogroup A, and occasionally, small outbreaks have been caused by serogroup
X isolates (4). However, sporadic cases of meningitis
caused by serogroup N. meningitidis W135 have been laboratory-confirmed
in Niger since the beginning of the 1980s (5). Strains
of serogroup W135 were found as often as strains of serogroup A at the
end of the epidemic of 2001 (6). In 2002 and again in
2003, Burkina Faso experienced the first large epidemics caused by N.
meningitidis serogroup W135 in Africa (7,8). This
event is a cause of concern for public health authorities of neighboring
countries, including Niger, and for the World Health Organization because
current affordable meningococcal vaccines available in large amounts target
only N. meningitidis serogroups A and C and do not protect against
serogroup W135. On the other hand, vaccines including the N. meningitidis
serogroup W135 valency are not only expensive but also rare, and this
situation will likely continue for the next 3 to 4 years. Enhanced microbiologic
surveillance is needed to quickly identify the serogroup of N. meningitidis
involved so that the appropriate vaccine for emergency mass vaccination
can be selected (9).
In Niger, few laboratories are able to perform etiologic diagnosis of
bacterial meningitis, although the country is large (1,267,000 km2)
and the population is scattered. Until 2002, microbiologic surveillance
existed but was inadequate because it focused almost entirely on the capital
city. The Centre de Recherches Médicales et Sanitaires (CERMES) in Niamey
became, in 2002, a national biomedical research center under the authority
of the Ministry of Health (MOH) and the national reference center for
meningitis in Niger. The polymerase chain reaction (PCR) method for the
diagnosis of acute bacterial meningitis was transferred from the Institut
Pasteur (Paris, France) for research purposes in October 2002. Therefore,
CERMES decided to include the PCR assay in the national framework of the
routine surveillance system of the MOH in November 2002 to be ready when
the next meningitis season began in January. The MOH has asked physicians
and nurses to systematically keep frozen (or at least refrigerated in
small health facilities) every sample of cerebrospinal fluid (CSF) collected
from patients with suspected cases of acute meningitis. Most often, in
remote healthcare centers and in the absence of laboratories, the clarity
of the CSF was assessed by macroscopic examination only, so the entire
volume of CSF that was previously discarded was kept for PCR analysis.
In the few district hospitals that have appropriate laboratory facilities,
the remaining CSF samples were stored after the latex agglutination assay
or bacteriologic tests had been performed. After laboratory personnel
had been informed of the purpose of the surveillance, sterile tubes and
epidemiologic forms were set up in 14 health districts within a radius
of approximately 250 km of Niamey. The designated area represented approximately
35% of the whole population. Afterwards, the CSF samples were regularly
collected by a CERMES vehicle, according to a precise timetable. Later,
tubes and forms were also set up in all health regions of Niger, and the
healthcare centers located beyond the limit of 250 km used any suitable
opportunities to convey the CSF samples to CERMES. Thus, the microbiologic
surveillance included >50% of the population. The implementation of
this strategy and the use of the results were carried out in close collaboration
with the national surveillance system.
CSF specimens were tested by PCR (amplification for 35 cycles) for the
three main causative agents of acute bacterial meningitis in Niger: N.
meningitidis (10), Streptococcus pneumoniae
(11), and Haemophilus influenzae (12).
A second PCR was performed on specimens positive for N. meningitidis
to identify serogroups A, B, C, and Y/W135, and finally, serogroups Y/W135
were differentiated from both Y and W135 alone by a confirmative PCR (10).
In the same manner, specimens positive for H. influenzae were further
tested for type b DNA.
From November 2002 to May 2003, which included the entire season of transmission,
1,651 CSF specimens collected within the national surveillance system
were processed by using PCR; 1,239 (75%) specimens came from outside the
capital, Niamey. Until mid-2002, most of these specimens would have been
lost for microbiologic surveillance. A total of 778 specimens (47.1%)
were positive: 661 for N. meningitidis (85%), 83 for S. pneumoniae
(10.1%), and 34 for H. influenzae (4.4%). The results of CSF examinations,
according to the month and region, are presented in Figures 1 and 2. Surveillance
highlighted that the meningococcal serogroup N. meningitidis W135
accounted for 8.5% of all N. meningitidis and that W135 was found
in most of the regions in Niger, although it did not cause epidemics.
N. meningitidis serogroup W135 was rare or absent in regions where
N. meningitidis serogroup A epidemics occurred (three serogroup
W135 isolates among 368 N. meningitidis isolates in Zinder and
no W135 among 79 N. meningitidis isolates in Maradi). With 284
specimens from Niamey, which had undergone both PCR and bacteriologic
testing, we compared the results from the two methods. The results of
both tests were in agreement for 231 specimens (81.3%); results for 182
of those specimens were negative. PCR found 25 positive samples for which
bacteriologic tests were negative, and 8 more positive specimens on which
bacteriologic testing could not be carried out because of contamination.
Conversely, bacteriologic testing provided a diagnosis for six PCR-negative
samples. However, the region of Niamey presented the lowest overall rate
of confirmation of suspected bacterial meningitis so this comparison is
less conclusive. The two diagnostic methods were also applied to 102 specimens
sent on trans-isolate medium. As is frequently observed, a contamination
problem occurred, and 32 samples (31.3%) were unsuitable for bacteriologic
testing, but PCR identified a causative agent in 20 of these 32 cases.
Of the remaining samples, for 46 (65.7%), bacteriologic testing and PCR
results were concordant, whereas 19 samples negative by bacteriologic
testing, tested positive by PCR. The opposite was observed for two specimens.
The overall proportion of specimens that tested positive for one of the
three species of bacteria varied according to the period. From November
to February, before the meningitis epidemic season, the positivity rate
was low in all the regions. In March and April, during the epidemic season,
the positivity rate remained low in the districts not undergoing epidemics,
although the positivity rate reached 60.9% in the Zinder region, where
epidemics due to N. meningitidis serogroup A occurred. Because
of the uncertainty of the cold chain in remote areas, some specimens may
have been stored without taking into account the temperature conditions,
which might have affected the sensitivity of the PCR testing. An analysis
of confirmation rates must also take into account that CSF samples were
tested whether they were cloudy or purulent or not and that the currently
used PCR focuses on three species only, although many other causes of
meningitis exist. The proportion of negative results may also indicate
that the national health services keep a vigilant watch over meningitis
and that the diagnosis is widely used, perhaps excessively. The symptoms
are not fully specific, and the predictive value of the clinical picture
for meningococcal meningitis increases substantially during an epidemic,
as shown by the results from Zinder.
In contrast to the culture method, which requires that laboratories receive
live bacteria (quite restricting, given the fragility of N. meningitidis),
PCR offers the substantial benefit of being able to be performed on dead
bacterial cells that have been killed by either refrigeration or previous
antimicrobial drug treatment. The simple way of storing and dispatching
CSF samples collected in remote areas was convenient and realistic for
the circumstances in Niger. Although the biologic confirmation by PCR
is retrospective in our study and cannot be used for case management,
this surveillance network compensates efficiently for the lack of functional
laboratories at the local level outside of the capital. During the 2003
meningitis season, PCR assay allowed satisfactory monitoring of the causative
agents of bacterial meningitis and of the involved meningococcal serogroups,
which is important in adapting the most appropriate preventive strategy
while serogroup W135 has the potential to cause epidemics in the countries
of the meningitis belt.
In conclusion, to compensate for the severe shortage of laboratories
outside the capital, the PCR assay proved to be a valuable tool for routine
microbiologic surveillance of bacterial meningitis in Niger. The country
has started implementing the Integrated Disease Surveillance and Response
plan within which this microbiologic surveillance is fully integrated.
Ms. Sidikou is a
biological engineer and the head of the molecular biology laboratory
in the Centre de Recherches Médicales et Sanitaires (CERMES) in Niamey,
Niger. Her major research interest is the enhancement of the microbiologic
surveillance of bacterial meningitides.
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