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Emerging Infectious Diseases
Volume 2, Number 2, April-June 1996

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Commentary

Molecular Epidemiology of Pneumocystis carinii Pneumonia

Pneumocystis carinii pneumonia (PCP) was first recognized as a distinct
clinical entity in orphanages in Europe during World War II (1). Today it
is the most frequent serious opportunistic infection in AIDS patients.
Despite advances in research, numerous questions remain regarding the basic
biology and epidemiology of P. carinii.

Transmission and Patient Care

Although reactivation of latent infections has long been considered the
primary explanation for PCP in immunosuppressed patients, over the years a
steady flow of reports has described clusters of PCP cases (2). In
addition, recent studies have suggested that the duration of latency is
very limited, i.e., usually less than 1 year (3,4). Still other studies
have demonstrated genetic variation in PCR-amplified P. carinii DNA from
the lungs of patients during subsequent PCP episodes (5). Together, these
observations provide strong circumstantial evidence of person-to-person
transmission of P. carinii. Consequently, establishing the role that
person-to-person transmission plays in the epidemiology of PCP is urgent.

Another important area of PCP epidemiology is determining the predisposing
factors for disease. The most frequently discussed predictor of disease is
CD4+ cell count, specifically as it relates to care and management of AIDS
patients (6); however, it has long been known that malnutrition can be an
important contributor (7). The degree to which other factors such as viral
infections or pneumonitis of other causes, may come into play, is yet to be
shown.

Much can also be learned regarding the epidemiology of PCP in HIV-infected
infants. Recent studies report that primary infections in these infants
often develop when the child is 3 to 6 months old (8,9). The source of
these patients P. carinii infections (i.e., the hospital setting, their
mothers, other children, or an environmental source) is not known.

Clinicians working with AIDS patients need a sensitive, reliable, and
noninvasive tool for early detection and diagnosis of PCP infections
(10,11). Besides the standard procedures of bronchoalveolar lavage (BAL)
and induced sputum (IS) sampling, recent studies indicate that it is
possible to amplify P. carinii DNA sequences by polymerase chain reaction
(PCR) directly from blood or serum samples and from nasopharyngeal
aspirates of PCP patients (11,12). Further studies are needed to confirm
that a PCR-based diagnostic tool superior to microscopy can be adapted for
use in clinical settings. A serologic tool that will distinguish recent PCP
infections from those past is also needed.

Prophylaxis failures have been reported for both
trimethoprim-sulfamethoxazole (TMP-SMX) and pentamidine (13-16). Studies
evaluating these cases, however, are frequently complicated by the
difficulties in assessing and confirming patient compliance with the
prophylaxis regimen. The only factor that has a significant correlation
with failure in most cases, however, is the patients CD4+ T lymphocyte
count (14). Although this correlation would be expected because of the
general increased risk for PCP associated with CD4+ cell depletion (6) and
the increase in prophylaxis complications in HIV-infected patients (17),
these drugs may not eliminate all organisms, and some degree of patient
immunity may be required to clear or control the infection. What role, if
any, specific antimicrobial resistance mechanisms play in the reported
treatment failures has not been shown; however, the emergence of resistance
is always a threat. Likewise, long-term TMP-SMX prophylaxis increases the
possibility for the selection of antimicrobial resistance in bacterial
pathogens, some of which are important potential causes of pneumonia in
HIV-infected patients (18). Identifying potential antimicrobial resistance
mechanisms in P. carinii is difficult because of the lack of an established
culture system for human P. carinii that would allow traditional
antimicrobial sensitivity testing.

At least three separate lines of data suggest that P. carinii is a commonly
encounteredorganism: the high seroprevalence rates reported in normal
populations (19), the rapid rate at which infants acquire primary
infections (8) and AIDS patients become reinfected after successful
treatment (20), and the amplification of P. carinii-specific DNA from
ambient air sampled from the environment (e.g., an apple orchard) (21) and
from rooms of animals and patients with PCP (22). Airborne transmission has
been demonstrated for PCP in rats (23-26) and is by far the most likely
mechanism proposed for natural exposure to P. carinii in humans (2,22).
Given the similarities between P. carinii and various fungal agents and the
enigma surrounding the issue of environmental sources for P. carinii, it
has been suggested that P. carinii may in fact be a dimorphic fungus,
ubiquitous in the environment and disseminated by airborne spores (27).
Identifying the specific environmental source or sources of P. carinii is
critical to understanding the epidemiology of PCP and establishing
guidelines for preventing its transmission.

It is generally accepted that P. carinii strains from rats do not infect
humans and that human strains do not infect rats; however, we do not know
the host boundaries for a given P. carinii strain or if all isolates from a
given host display the same degree of host restrictions (28,29). In fact, a
careful evaluation of the available data concerning P. carinii of numerous
hosts suggests that P. carinii may represent a collection of diverse fungal
species (30). Like drug resistance research, studies aimed at
strain/species characterization are generally hindered by the difficulties
in culturing human P. carinii and the lack of refined molecular biological
methods that allow strain identification and characterization.

Molecular Biologic Techniques and Specific Epidemiologic Issues

One of the essential reasons for cultivating any particular pathogen is for
strain identification and characterization that would elucidate such
specific phenotypic characteristics as virulence factors, antimicrobial
sensitivity levels, and factors associated with transmissibility. The
isolation and cultivation of individual strains, and ultimately of clones,
would provide a homogeneous population of organisms from which the desired
information can be obtained and a pure source of genetic material for
constructing DNA libraries and identifying relevant genes.

In the absence of cultivation, investigators have been able to begin
addressing some of the basic epidemiologically important issues by applying
PCR-based technology. In these studies, the DNA sequence of specific
genetic loci from P. carinii is usually amplified from BAL, IS, or serum
samples from PCP patients, using highly specific oligonucleotide primers.
Inherent problems exist in this approach (which are discussed below);
however, the approach has allowed the identification of genotypic
differences in P. carinii populations sampled from the lungs of different
patients and even from the lungs of the same patient during different PCP
episodes. Great potential exists in applying this technology to develop
molecular profiles of P. carinii isolates that could ultimately allow the
particular genotypes to be linked to specific epidemiologically relevant
phenotypes.

Molecular Typing

Five to ten different genetic loci have been identified as potentially
informative for molecular characterization and typing (30-33). Concerning
the typing that has actually been performed on human samples, the primary
loci evaluated include: 1) a 346-bp region of the mitochondrial large
subunit rRNA gene (mt lsrRNA) (10) and 2) a 550-bp fragment containing the
nuclear ribosomal internal transcribed spacer regions 1 and 2 (ITS1 and
ITS2) (34). When these loci are considered collectively, nucleotide
variation can be detected at approximately 37 different positions. Work is
in progress in several laboratories, both to type patient isolates
according to the available loci and to identify additional genetic loci to
more thoroughly define a given genotype.

The primary obstacles to the development of a molecular typing scheme based
on PCR-amplified DNA sequence data obtained from PCP patients include the
following: 1) multiple strains may infect a single patient at a given time;
2) a diploid organism of a single strain may be heterozygous with respect
to a particular polymorphic locus; 3) presumed single genes could have
multiple copies in a single genome, which could give the appearance of
genetic polymorphism; 4) amplified DNA sequence data might be confounded
because of other fungal agents such as Cryptococcus or Candida; and 5)
inferences that can be drawn from restricted sequence data (i.e., gene
typing versus strain typing) are limited. Although these problems are not
insurmountable, they must be considered when evaluating data obtained by
this approach. We propose the following recommendations.

Recommendations

In September 1995, the U.S. Centers for Disease Control and Prevention
hosted a meeting to discuss issues relating to molecular biologic
approaches for studying the epidemiology of P. carinii pneumonia. The
meeting was attended by representatives of research laboratories and by CDC
personnel involved in the epidemiology and molecular biology of P. carinii.
These recommendations regarding research priorities emerged from this
meeting.

Molecular Epidemiology

1. Recent advances in molecular-based typing should be combined with
epidemiologic studies to investigate the transmission of P. carinii and new
strategies for control.

2. Additional genomic regions must be identified for use in typing, along
with the genetic loci that are available. These new loci must be shown to
represent single-copy genes. Also, new molecular approaches should be
developed that will increase the current capacity to resolve genotypic
variation among P. carinii strains.

3. Genetic variation should be investigated among P. carinii strains that
could be linked to variations in factors such as strain virulence, drug
resistance, or transmissibility.

4. The critical issue regarding person-to-person transmission is not so
much whether it occurs, as whether it contributes to infection
significantly more than airborne sources in the environment. Thus, it must
be determined whether there is any benefit to establishing complex
protocols that ensure that patients are carefully protected from each other
if they can become infected from other sources in the environment.
Consequently, the importance of person-to-person transmission in the
epidemiology of PCP should be defined.

5. The role of latent P. carinii infection as a source of PCP in
immunocompromised persons should be clarified.

Diagnosis, Treatment, and Prevention

1. New tools for noninvasive early diagnosis of PCP, including culture
systems, molecular approaches, and serologic tests that can distinguish
recent and past PCP infections are needed.

2. In the United States, clinician compliance with recently published U.S.
Public Health Service/Infectious Diseases Society of America guidelines on
the treatment and prophylaxis of PCP should be evaluated.

3. Studies should be initiated to develop additional drugs for PCP
treatment and prophylaxis.

4. New approaches for improving patient compliance with prescribed PCP
prophylaxis must be devised and evaluated.

5. Methods for detecting the possible emergence of drug resistance to P.
carinii should bestandardized.

6. Standard decontamination procedures for respiratory therapy equipment
and pulmonary diagnostic instruments should be evaluated to confirm that
they effectively eliminate all viable P. carinii.

Environmental Reservoirs and General Biology

1. Environmental sources and the coinciding infective stage(s) of P.
carinii should be detected and evaluated.

2. The host range of P. carinii from various sources (i.e., to what degree
are humans susceptible to P. carinii from nonhuman sources?) should be
determined.

Acknowledgments

We thank the following colleagues for their contributions: C. William
Angus, Marilyn S. Bartlett, Jeanne M. Bertolli, Suzanne Binder, Daniel G.
Colley, Frank H. Collins, Melanie T. Cushion, Alexandre J. DaSilva, Jeffrey
S. Duchin, Brian R. Edlin, Walter T. Hughes, John A. Jernigan, Dennis D.
Juranek, Jonathan R. Kaplan, Scott P. Keely, Chao-Hung Lee, Joseph E.
McDade, Stephen A. Moser, Norman J. Pieniazek, Frank F. Richards, James W.
Smith, Eleanor K. Spicer, James R. Stringer, and Craig M. Wilson. Mary E.
Bartlett provided editorial assistance.

Charles B. Beard, Ph.D., and Thomas R. Navin, M.D.
Centers for Disease Control and Prevention
Atlanta, Georgia, USA

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Commentary

Needed: Comprehensive Response to the Spread of Infectious Diseases

In his article Globalization, International Law, and Emerging Infectious
Diseases, Fidler recognizes that biological agents travel by themselves or
with their hosts without any recognition of, or regard for, political
borders. He notes that with the continued expansion of economic commerce
across continents and more rapid transport and travel, persons infected
with infectious diseases of very short incubation periods can act as
vectors across several nations before they even become symptomatic. The
protective effect of clipper ship travel is long gone.

Fidler examines the need for international treaties, agreements, and
policies to manage the spread of new or reemerging infections diseases. His
concern is that the current international climate requires more enforceable
treaties with adequate resources to identify, track, interfere with, and
contain the spread of infectious diseases perceived as an international or
global threat.

International cooperation within the existing legislative mechanisms has,
on occasion, been very successful. International eradication of smallpox
was successful because a specific, cost-effective, efficient vaccine was
developed; the disease attacked persons regardless of their economic,
political, racial, religious, or social affiliations; the amount of funding
was adequate; and all nations recognized the benefits of the eradication
program. A similar effort currently in progress to eradicate poliomyelitis
will also be successful because of international cooperation.

In contrast, international control of other infectious diseases, such as
malaria and tuberculosis, has been attempted for decades with considerably
less success. Notwithstanding the lack of efficacious vaccines, the reality
is that only very limited resources are being committed to prevent and
treat all infectious diseases. Outbreaks of Ebola virus infection and
plague are routinely reported in the local, national, and international
press. However, the continued increased incidence and prevalence of
tuberculosis, AIDS, and other sexually transmitted diseases are accepted by
many as problems of the poor, the immoral, and the expendable portion of
society. Local, national, and international awareness and continued
interest are significant problems.

International cooperation must extend beyond merely restricting the natural
spread of specific diseases. One also has to recognize the need for
effective international treaties to prevent the use of biological agents in
either tactical or strategic circumstances. Fear of combatants using
biological agents on military and civilian targets intensified during and
since the Gulf War. The possibility of biological terrorism is no longer
limited to the imagination of fiction writers. Fidler does not stress the
issue of nonnatural outbreaks of diseases; a global need for an improved
non-ad hoc response to emerging infectious disease agents should be
completely considered by civilian and military planners. The threat of
infectious diseases as weapons provides an additional incentive for
cooperation among governments.

Harold M. Ginzburg, M.D., J.D., M.P.H.
Department of Psychiatry and Neurology, Tulane University Medical Center,
New Orleans, Louisiana, USA